CHARACTERIZATION OF WINTER SQUASH AGE-RELATED RESISTANCE TO PHYTOPHTHORA CAPSICI THROUGH FRUIT PEEL TRANSCRIPTOME PROFILING AND INVESTIGATION OF CELL WALL PROPERTIES By Safa Abdelghaffar Alzohairy A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Plant Breeding, Genetics and Biotechnology-Crop and Soil Sciences- 2018 ABSTRACT CHARACTERIZATION OF WINTER SQUASH AGE-RELATED RESISTANCE TO PHYTOPHTHORA CAPSICI THROUGH FRUIT PEEL TRANSCRIPTOME PROFILING AND INVESTIGATION OF CELL WALL PROPERTIES By Safa Abdelghaffar Alzohairy Fruit rot of winter squash and pumpkin (Cucurbita moschata) caused by the oomycete plant pathogen Phytophthora capsici limits factor in the production of these crops. Genetic resistance to fruit rot is not available in commercial cultivars, but age-related resistance (ARR) develops in certain cultivars of C. moschata which may benefit management strategies. Earlier ARR studies indicate that the peel provides fruit resistance. The goal of this research was to elucidate the structural, biochemical and genetic basis of ARR of the winter squash fruit peel to P. capsici. Five C. moschata cultivars were evaluated for P. capsici resistance 10, 14, 16, 18, and 21 days post pollination (dpp). The onset of resistance was variable among cultivars. A cultivar with ARR at 14 dpp was selected and the fruit exocarp cell wall examined 7 dpp (susceptible) and 14 dpp, 21 dpp (resistant) using scanning electron microscopy (SEM). An increase in cuticle and epidermal walls thickness as the fruit age increased was observed. According to SEM observations, P. capsici caused cell wall degradation/tissue collapse to the 7 dpp fruit within 48- hour post inoculation (hpi) while 14 and 21 dpp fruit remained unaffected suggesting a structural barrier to P. capsici in resistant fruit. The contribution of fruit exocarp preformed or induced chemical defense against the pathogen was examined in C. moschata cultivars across developmental stages using phytochemical analysis. Results showed a decrease in antifungal activity in non-inoculated fruit peel as the fruit age increased. A significant change in antifungal activity was not observed under induced conditions with inoculation of the fruit peel with P. capsici, suggesting that there is not a correlation between preformed or induced chemical defense and winter squash fruit ARR to P. capsici. Transcriptome profiling of fruit peel of two C. moschata cultivars at susceptible and resistant developmental stages was performed to uncover the molecular mechanism of ARR. Differential gene expression analysis detected upregulation of multiple genes in the resistant compared to the susceptible stages then functional enrichment analysis detected overrepresentation of these genes in cell wall structures biosynthesis. Pathway enrichment analysis of winter squash orthologous genes detected enrichment in cutin, suberin monomers and phenylpropanoids biosynthetic pathways. Further analysis of genes expression profile in those pathways suggests enrichment in monolignol biosynthesis in the resistant fruit peel. I cannot ever find enough words to thank you for being a good example in my life. To my parents; Love you so much. iv ACKNOWLEDGEMENTS I am very grateful to my supervisor and great mentor Dr. Mary Hausbeck for believing in me and giving me the opportunity to succeed. Thank you for all you help, compensations, and encouragement that you gave me through my PhD. I will never forget your favor that was the first step in my success, achieving my dream in pursuing my PhD and making my career in plant pathology. I am very thankful to my committee members for their time and directions. Many thanks to Dr. Raymond Hammerschmidt for giving me unlimited support and teaching me different techniques in physiology and biochemistry. It was a great honor for me to work with you and learn from your expertise. I really enjoyed and learned a lot from our discussions. I will never forget that your lab was my second lab to finish my research experiments and will never forget your help and encouragement. To Dr. Shinhan Shiu, I am very thankful to all your help that you gave me since I started my PhD at MSU. Thanks for your patient in teaching me the critical thinking and scientific writing skills. I am very thankful for the all the time that you have spent to help me achieving my goals and encouraging me to pursue my dreams. I am very thankful to Dr. Cholani Weebadde, for the valuable discussion and directions that you gave me through my PhD and for your unlimited encouragement and being excited about my research. I would to thank all Hausbeck’s lab members. I am very thankful to Dr. Charles Krasnow for teaching me the field work and fruit disease assessment. Special thanks to Dr. Beth Brisco, Dr. Yufang Guo, Irene Donne, Doug Higgins, Sara Getson, David Perla, Julian Bello, Katelyn Goldenhar, Alex Cook, Matt Bour, Balir Harlan, Sheila Linderman. I am very appreciated to all help that you gave me through my academic journey. I would like to thank John Baltusis, the v former undergraduate who has helped me in my project especially with the field work. It was very nice to work with a responsible person like you. I would like to thank all the Hammerschmidt’s lab members for their help through my work in their lab and helping me finishing my experiments. Special thanks to Dr. Linzi Kaniszewski who taught me the TLC bioassay technique and for her encouragement and lovely friendship. I would like to thank all my friends in plant pathology and plant breeding, genetics and biotechnology programs for their help and support through my academic journey. To all Plant Soil and Microbial Sciences, Plant Pathology office members, I am very thankful to all your help through my academic journey. I would like to thank all my sisters and brothers from the East Lansing Islamic Center who provided me with all help to make my life easier while having kids and working for my PhD. Finally, but not last, thanks to my husband Hussien Alameldin for his thoughtful and sincere character and believing in me. Thanks for every moment that you spent helping me in my work and life. Thanks for making my life with having kids and working for pursuing my PhD easier. Truly there are no enough words to tell you thanks for everything you made to help me achieving my dream and be a successful woman. Thanks for our three boys Mohammad, Ahmad, and Ali Alameldin, the three gifts of my PhD. vi TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES ........................................................................................................................ x LITERATURE REVIEW ............................................................................................................... 1 PATHOGEN BIOLOGY ............................................................................................................ 3 INFECTION PROCESS ............................................................................................................. 4 PATHOGEN HOST RANGE AND SYMPTOMS .................................................................... 5 DISEASE MANAGEMENT STRATEGIES ............................................................................. 6 HOST RESISTANCE TO P. CAPSICI ...................................................................................... 7 THE GENETIC BASIS OF RESISTANCE ............................................................................. 12 CONCLUSION ......................................................................................................................... 13 LITERATURE CITED ............................................................................................................. 14 CHAPTER I: CHANGES IN WINTER SQUASH FRUIT EXOCARP STRUCTURE ASSOCIATED WITH AGE RELATED RESISTANCE TO PHYTOPHTHORA CAPSICI ...... 23 ABSTRACT .............................................................................................................................. 23 INTRODUCTION .................................................................................................................... 24 MATERIALS AND METHODS .............................................................................................. 27 RESULTS ................................................................................................................................. 31 DISCUSSION ........................................................................................................................... 41 LITERATURE CITED ............................................................................................................. 46 CHAPTER II: ANTIFUNGAL ACTIVITY IN WINTER SQUASH FRUIT PEEL IN RELATION TO AGE RELATED RESISTANCE TO PHYTOPHTHORA CAPSICI ................ 51 ABSTRACT .............................................................................................................................. 51 INTRODUCTION .................................................................................................................... 52 MATERIALS AND METHODS .............................................................................................. 54 Plant material ........................................................................................................................ 54 Phytophthora capsici inoculum ............................................................................................ 55 Non-inoculated fruits for the Thin Layer Chromatography bioassay ................................... 55 Preparation of inoculated fruits for Thin Layer Chromatography bioassay ......................... 55 Thin Layer Chromatography Bioassay Methodology ........................................................... 56 Bioassay ................................................................................................................................ 57 RESULTS ................................................................................................................................. 58 DISCUSSION ........................................................................................................................... 64 LITERATURE CITED ............................................................................................................. 67 CHAPTER III: TRANSCRIPTOMIC PROFILING OF WINTER SQUASH IMPLICATES MONOLIGNOLS BIOSYNTHESIS AND LIGNIN POLYMERIZATION IN AGE-RELATED RESISTANCE TO THE OOMYCETE PHYTOPHTHORA CAPSICI ........................................ 72 ABSTRACT .............................................................................................................................. 72 INTRODUCTION .................................................................................................................... 73 MATERIALS AND METHODS .............................................................................................. 75 vii Plant material ........................................................................................................................ 75 RNA extraction ..................................................................................................................... 76 RNA sequencing and RNA-seq read processing .................................................................. 76 Differential expression and clustering analysis .................................................................... 77 Identification of putative Arabidopsis orthologous genes and inference of squash cell well pathway genes ....................................................................................................................... 78 Functional annotation and pathway enrichment analysis ..................................................... 78 RESULTS ................................................................................................................................. 79 Sequencing and gene expression profile among cultivars .................................................... 79 Differential gene expression of ‘Chieftain’ and ‘Dickenson Field’ ...................................... 81 Function of upregulated genes in both cultivars ................................................................... 84 Function of downregulated genes in both cultivars .............................................................. 87 Comparison of ARR mechanism among cultivars ................................................................ 88 Pathway enrichment of cell wall structure-related genes ..................................................... 90 DISCUSSION ........................................................................................................................... 96 CONCLUSION ....................................................................................................................... 102 APPENDIX ............................................................................................................................. 103 LITERATURE CITED ........................................................................................................... 118 FUTURE RESEARCH ............................................................................................................... 125 viii LIST OF TABLES Table 1.1.: Cucurbita moschata with listed cultivars used in the study. ..................................... 28 Table 1.2.: Disease rating, lesion diameter and disease incidence four days post inoculation with P. capsici for 5 cultivars of Cucurbita moschata at fruit developmental stages 10, 14, 16, 18, and 21 days post pollination (dpp). ...................................................................................................... 32 Table 1.3.: Minimum and maximum thickness (µm) of cell wall structures: cuticle, epidermal anticlinal wall top-point, epidermal anticlinal wall mid-point, and cortex cell wall across fruit development at 7, 14, and 21 days post pollination (dpp) in cultivar Chieftain. .......................... 35 Table 1.4.: Pairwise comparisons of cell wall structures between fruit developmental stages of cultivar Chieftain. ......................................................................................................................... 35 Table 3.1.: Number of raw reads, alignment count, and uniquely mapped reads. ..................... 104 Table 3.2.: Gene Ontology enrichment of upregulated genes in the resistant stages in both cultivars resulted from all contrasts. The values are the negative log of the q-value if the GO term is overrepresented, or the log of the q-value if the GO term is underrepresented. At q = 0.05, a term is significantly overrepresented if the value is > or = to 1.3 and significantly underrepresented if the value is < or = to -1.3. ........................................................................... 105 Table 3.3.: Gene Ontology enrichment of downregulated genes in the resistant stages in both cultivars resulted from all contrasts. The values are the negative log of the q-value if the GO term is overrepresented, or the log of the q-value if the GO term is underrepresented. At q = 0.05, a term is significantly overrepresented if the value is > or = to 1.3 and significantly underrepresented if the value is < or = to -1.3. ........................................................................... 109 ix LIST OF FIGURES Figure 1.1.: Average disease rating of Cucurbita moschata cultivars’ fruits at 10, 14, 16, 18, and 21 dpp in response to Phytophthora capsici inoculation, four days post inoculation (disease rating 0 = no visible pathogen growth; 1 = water-soaked tissue only; 2 = light visible mycelial growth; 3 = moderate mycelial growth; and 4 = dense mycelial growth). .................................................... 33 Figure 1.2.: Scanning electron microscopy images of cross sections of non-wounded/non- inoculated fruits of cultivar Chieftain (a) 7 dpp, (b) 14 dpp, and (c) 21 dpp. Arrows for (c) represent the cell wall structures and the measurement direction where a: cuticle, b: epidermal anticlinal wall top-point, c: epidermal anticlinal wall mid-point, and d: cortex cell wall. Arrows heads represent the direction of measurement. ....................................................................................................... 34 Figure 1.3.: Histogram showing average thickness (µm) of cell wall structures: cuticle, epidermal anticlinal wall top-point, epidermal anticlinal wall mid-point, and cortex cell wall across fruit development at 7, 14, and 21 dpp of cultivar Chieftain. Same color column with different letter indicates significant difference (P £ 0.05) of cell wall structure thickness across ages. .............. 36 Figure 1.4.: Scanning electron microscopy images of transverse sections at non-wounded/non- inoculated fruits of cultivar Chieftain. (a) 7 dpp, (b) 14 dpp, and (c) 21 dpp. Wax appears as spiny crystals on fruit surface. ................................................................................................................ 36 Figure 1.5.: SEM images of cross sections at 6 hour post inoculation (hpi), 24 hpi, and 48 hpi of cultivar Chieftain fruits at (a, d, g) 7 dpp, (b, e, h) 14 dpp, and (c, f, i) 21 dpp, respectively, showing the effect of Phytophthora capsici inoculation on cell wall integrity. The white circles point for the inoculation site. ....................................................................................................................... 37 Figure 1.6.: Scanning electron microscopy images of cross sections of cultivar Chieftain fruits of 7 days post pollination (dpp) at (a) 6 hours post inoculation (hpi), (b) 48 hpi; 14 dpp at (c) 6 hpi, (d) 24 hpi, (e) 48 hpi; and 21 dpp at (f) 6 hpi, (g) 24 hpi, and (h) 48 hpi. Hy=hypha; c=cuticle; eaw=epidermal anticlinal wall; vb=vascular bundle. ................................................................... 38 Figure 1.7.: Scanning electron microscopy images of the top surface view of cultivar Chieftain fruits. (a) 7 days post pollination (dpp) at 6 hours post inoculation (hpi), hyphal direct penetration from the epidermal surface (b) 7 dpp at 6 hpi, hypha passing over stomata without entering, (c) 7 dpp at 6 hpi, hypha traveling toward entering through wound and bypassing multiple stomata; (d) 7 dpp at 24 hpi, hypha branching and directly penetrating; (e) 7 dpp at 48 hpi, multiple penetration points; (f, g) 14 dpp and 21 dpp, respectively, at 6 hpi showing hyphae passing over stomata but not entering; (h, i) 14 dpp and 21 dpp, respectively, at 24 hpi showing hyphae entering through stomata; sr=surface ridge; es=epidermal surface; hy=hypha; st=stomata; w=wound; ap= appressorium. ................................................................................................................................ 40 Figure 2.1.: Thin Layer Chromatography (TLC) bioassay of ‘Dickenson Field’ non-inoculated fruit peel methanol extracts at 10, 14, 21 days post pollination (dpp). Top images are methanol extract of three biological replicates, R1, R2, and R3 of ‘Dickenson Field’. The extracts were x applied to a cellulose plate and developed in distilled water. Lanes 1, 2, and 3 include 25mg of 10, 14, and 21 dpp respectively. Images were captured under UV light 365nm. Bottom images are the TLC plates sprayed with Cladosporium cucumerinum and incubated for 48hrs under humidity. White areas in the red boxes refer to zones of inhibition…………………………………………59 Figure 2.2.: Thin Layer Chromatography (TLC) bioassay of ‘Early’ (lanes 1 to 3), ‘Dickenson Field’ (lanes 4 to 6), and ‘Chieftain’ (lanes 7 to 9) non-inoculated fruit peel methanol extracts at 10, 14, 21 days post pollination (dpp) respectively. Top images are sample methanol extracts applied to a silica-G-gel plate and developed in chloroform-methanol (9:1). Images were captured under UV light 365nm. Bottom images are the TLC plates sprayed with Cladosporium cucumerinum and incubated for 48hrs under humidity. White areas in the red box refer to zones of inhibition. ...................................................................................................................................... 60 Figure 2.3.: Thin Layer Chromatography (TLC) bioassay of ‘Chieftain’ inoculated fruit peel at 7, 10, 14, 21 days pos pollination (dpp) with Phytophthora capsici at 6 hours control and 6 hours post inoculation (hpi). Top images are samples ethanol extracts applied to a silica-GF-gel plate and developed in chloroform-methanol (9:1). Lanes 1 – 4 include 50mg of 7, 10, 14, and 21 dpp at 6 hours control and lanes 5 – 8 include 50mg of 7, 10, 14, and 21 dpp at 6 hours post inoculation (hpi). Images were captured under UV light 365nm. Bottom images are the TLC plates sprayed with Cladosporium cucumerinum and incubated for 48hrs under humidity. White areas in the red box refer to zones of inhibition. .................................................................................................... 62 Figure 2.4.: Thin Layer Chromatography (TLC) bioassay of ‘Chieftain’ inoculated fruit peel at 7, 10, 14, 21 days post pollination (dpp) with Phytophthora capsici at 6 hours control and 6 hours post inoculation (hpi). Top images are samples ethanol extracts applied to a silica-GF-gel plate and developed in chloroform-methanol (9:1). Lanes 1 – 4 include 50mg of 7, 10, 14, and 21 dpp at 12 hours post inoculation (hpi) and lanes 5 – 8 include 50mg of 7, 10, 14, and 21 dpp at 24 hours post inoculation (hpi). Images were captured under UV light 365 nm. Bottom images are the TLC plates sprayed with Cladosporium cucumerinum and incubated for 48hrs under humidity. White areas in the red box refer to zones of inhibition. ........................................................................... 63 Figure 3.1.: A) Expression conditions of cultivars Chieftain and Dickenson fruit peel at ages 7, 10, 14, and 21 days post pollination. B) Heatmap of K-means clustering of per-gene normalized expression between both ‘Chieftain’ and ‘Dickenson’ at 7, 10, 14, 21 dpp. Numbers 1, 2, 3, and 4 are different groups of clusters. Genes were clustered at K=16………………………………….80 Figure 3.2.: Differential gene expression analysis of ‘Chieftain’ and ‘Dickenson’ fruits at 7, 10, 14 and 21 days post pollination (dpp). The onset of age-related resistance (ARR) is at 14 and 21 dpp in ‘Chieftain’ and ‘Dickenson’, respectively. A) Heatmap of differentially expressed genes (DEGs) with |log2(FC)|>1, (FC: fold change) and adjusted p-values <0.05. Each column is a contrast between resistant vs susceptible peel of fruit at 7, 10, 14 and 21 dpp. A) and B) Venn diagrams showing upregulated and downregulated genes in all sets of comparisons in ‘Chieftain’ respectively; C) and D) Venn diagrams showing upregulated and downregulated genes in all sets of comparisons in ‘Dickenson’, respectively. The letters C and D indicates ‘Chieftain’ and ‘Dickenson’, respectively. ............................................................................................................ 82 xi Figure 3.3.: Heatmap showing selected GO terms that are significantly overrepresented in either upregulated genes or downregulated genes in contrasts between resistant vs susceptible peel of fruit at 7, 10, 14 and 21 days post pollination (dpp) in both ‘Chieftain’ and ‘Dickenson’. The onset of age-related resistance (ARR) is at 14 and 21 dpp in ‘Chieftain’ and ‘Dickenson’ respectively. The letters C and D indicates for ‘Chieftain’ and ‘Dickenson’ respectively. The value range of the heatmap is shown as the result of the Fisher’s Exact test. If the GO term was overrepresented, the negative log of the adjusted p-value (or q-value) was taken, while if it was underrepresented, the log of the adjusted p-value was taken. Therefore, a positive value > or = 1.3 indicates significant overrepresentation while a negative value < or = -1.3 indicates significant underrepresentation. The black rectangles point to functions related to cell wall structures and phenylpropanoid biosynthesis processes that are overrepresented only in the upregulated genes in both cultivars. 85 Figure 3.4.: Heatmap showing the pathway enrichment analysis of hard squash of C. moschata orthologous genes in Arabidopsis cell wall structures biosynthetic pathways. Columns show the contrast between resistant vs susceptible peel of fruit at 7, 10, 14 and 21 days post pollination in both ‘Chieftain’ and ‘Dickenson’. The onset of age-related resistance (ARR) is at 14 and 21 in ‘Chieftain’ and ‘Dickenson’ respectively. The letters C and D indicates for ‘Chieftain’ and ‘Dickenson’ respectively. The value in the range of the heatmap is shown as the results of the Fisher’s Exact test. If the pathway was overrepresented, the negative log of the adjusted p-value (or q-value) was taken, while if it was underrepresented, the log of the adjusted p-value was taken. Therefore a positive value > or = 1.3 indicates significant overrepresentation while a negative value < or = -1.3 indicates significant underrepresentation. .......................................... 91 Figure 3.5.: General expression profile of squash orthologous genes that are differentially expressed (p<0.05, Log2(FC) >1, FC: Fold Change) in the different contrasts in both cultivars and involved in A) phenylpropanoid, B) suberin monomers, C) cutin, D) cellulose, and E) homogalacturonan biosynthetic pathways. ................................................................................... 93 Figure 3.6.: Expression profile of squash orthologous genes that are differentially expressed (p<0.05, Log2(FC) >1, FC: Fold Change) in the different contrasts in both cultivars. A, B, C) DEGs involved in cutin, suberin monomers, and phenylpropanoid biosynthetic pathways respectively. .................................................................................................................................. 95 xii LITERATURE REVIEW Cucurbitaceae is a large, diverse family that includes approximately 825 species across 118 genera (Bisognin 2002). The Cucurbitaceae family contains many vegetable crops commonly known as cucurbits. Cucurbits are mostly monoecious, tendril-bearing climbing plants, common in tropical and subtropical areas of Africa, Asia, Australia, and America. They prefer warm temperatures and are frost-sensitive but are found in temperate regions (Crase 2011). Cucurbits include crops grown for food and ornamentation. Some cucurbits have medicinal uses due to the compound Cucurbitacin that has anti-inflammatory properties (Abdelwahab et al. 2011). The most important genera of this family are Cucumis (includes cucumber and melons), Citrullus (includes watermelon), and Cucurbita (includes squash, pumpkins, and some gourds) (Whitaker and Davis 1962). Cucumber originated from India and melon and watermelon originated from Africa. Squash was originally discovered in South and Central America (Wehner et al. 2003). The Cucurbita genus is one of the most morphological diverse genera of the plant kingdom (Robinson et al., 1976). It includes 22 wild and five cultivated species including C. pepo L., C. maxima Duchesne, C. moschata Duchesne, C. argyrosperma Huber and C. ficifolia Bouché, with diverse characteristics in color, shape, and size (Bisognin 2002). Cucurbita pepo includes pumpkin, gourd, acorn squash, summer squash, and zucchini. C. moschata includes winter squash and pumpkin. C. maxima produces the largest fruit among the flowering plants. Fruits of these crops are eaten when immature (C. pepo) or mature (C. maxima and C. moschata) (Bisognin 2002). Worldwide, these fruits are known as a rich source of vitamin A that is important for human health (Shrivastava et al. 2013). Fruits can be baked as a main dish or served as a dessert, and their seeds toasted providing a source of protein and oil (Wehner et al. 2003). Flowers, leaves, and vine tips are also consumed in regions including Latin America (Ferriol and Pico 2008). The cultivated 1 species C. argyrosperma and C. ficifolia are not widely distributed and are not economically important. Squash and pumpkins are grown worldwide for both the fresh market and for processing. China is the top producer of squash followed by India, Russia, and the U.S. (FAO 2012). In the U.S., Michigan leads the country for zucchini, summer and hard squash production for fresh market and processing followed by Florida, California, and New York (USDA 2018). In 2017, squash was grown on 5,800 acres in Michigan generating more than $26 million (USDA 2018). The most popular types of squash grown in Michigan include summer squash (yellow and scallop) and zucchini, and the winter squashes (butternut, buttercup, marrow, and acorn) (Pollack 1996). Michigan ranks 4th for pumpkin production following Illinois, Ohio, and Indiana. In 2017, pumpkins were grown on 5,300 acres in Michigan and valued at more than $8 million (USDA 2018). In Michigan, summer and zucchini squash are direct seeded in late April through late May with harvest beginning in mid-June. Winter squash planting occurs at the same time and continues through early June with harvest beginning in early September (USDA 2018). Due to their monoecious nature, squash is reliant on bee and insect pollination for complete fertilization and fruit set. Summer squash and zucchini are harvested as immature fruit, and winter squash is harvested when the fruits are fully mature and ripened with a hard rind. Fruit rot caused by Phytophthora capsici Leonian (Leonian 1922) limits the production of cucurbits. This disease also threatens fruiting vegetables including tomato, pepper, and eggplant (Tian and Babadoost 2004; Hausbeck and Lamour, 2004). Cucurbits affected by this pathogen include squash, watermelon, cucumber, pumpkin, cantaloupe, gourd, and honeydew. P. capsici 2 causes symptoms including crown rot, foliar blight, and/or fruit rot (Roberts et al., 2001; Babadoost 2005). PATHOGEN BIOLOGY Phytophthora capsici classifies as a eukaryote in kingdom Stramenopila (Cavalier-Smith 1986), family Pythiaceae, order Peronosporales and class Oomycetes. The pathogen is producing oospores and biflagellate zoospore (Erwin and Ribeiro 1996, Hardham 2007, Mrázková et al. 2011). The cellulose in its cell wall differentiates it from fungi (Hardham 2007, Erwin and Ribeiro 1996). P. capsici is heterothallic, requiring two mating types, A1 and A2, for sexual reproduction (Erwin and Ribeiro, 1996, Lamour and Hausbeck 2000). When both mating types coexist in the same field, the mating types produce specific hormones that stimulate the production of gametangia then outcrossing occur and oospores are produced (Ko 1988). Oospores have a thick- wall containing β-glucans and cellulose (Erwin and Ribeiro 1996, Bartnicki-Garcia and Wang 1983), which aids pathogen survival in harsh environmental conditions. Oospores can remain viable in the field for a prolonged period (Erwin and Ribeiro 1996, Lamour and Hausbeck 2000, Hausbeck and Lamour 2004, Babadoost and Pavon 2013). Oospores germinate to produce a germ tube and stimulated by root exudates and natural chemicals produced by the plant (Erwin and Ribeiro 1996, Hord and Ristaino 1991). The germ tube can either elongate to form hypha or develop sporangia (Hord and Ristaino 1991). During the asexual stage, P. capsici produces aseptate hyphae that grow at an optimum temperature of 24 – 33oC and produce papillate sporangia with a long pedicle (Babadoost 2004). P. capsici sporangia can be subspherical, ovoid, obovoid, ellipsoid, fusiform, or pyriform (Babadoost 2004) with variation influenced by light or other environmental factors (Erwin and 3 Ribeiro 1996, Babadoost 2004). Mature sporangia are caducous and depend on water for dissemination (Granke et al. 2012). P. capsici sporangia can germinate to produce a germ tube and infect directly (Judelson and Blanco 2005), or under saturated conditions, the sporangial cytoplasm differentiates into 20 – 40 biflagellate swimming zoospores (Bernhardt and Grogan 1982, Hausbeck and Lamour 2004, Granke et al. 2012). The zoospores are negatively geotropic and are chemotactically attracted to the plant host following the nutrient gradient (Khew and Zentmyer 1973, Erwin and Ribeiro 1996). INFECTION PROCESS When zoospores come into contact with a plant’s surface, they encyst within 3 hours (Du et al. 2013) and produce a germ tube (Hickman 1970). P. capsici secretes cell wall degrading enzymes that aid in the penetration of the cuticle (Yoshikawa et al. 1977, Jia et al. 2009). The mechanism of P. capsici hyphal penetration is direct or through natural opening such as a wound or stomata (Judelson and Blanco 2005, Katsura and Miyazaki 1960). However, Du et al. (2013) showed no penetration through stomata even when P. capsici hypha passed over it. Formation of appressoria was observed in some cases, while not in others (Lamour et al. 2012, Du et al. 2013). P. capsici is a hemibiotroph, the biotroph stage occurs during the 2 to 3 days from infection to sporulation. The necrotrophic stage occurs when clusters of mycelia develop inside the tissue and colonize resulting in damage and maceration of the host tissue (Lamour et al. 2012, Du et al. 2013). P. capsici is a polycyclic pathogen with multiple disease cycles. Thus, even a low amount of inoculum may initiate a significant disease outbreak (Erwin and Ribeiro 1996, Hausbeck and Lamour 2004). When environmental conditions are favorable, the pathogen produces sporangia on the surface of infected tissue (Hausbeck and Lamour 2004, Granke et al. 2009). The number 4 of sporangia produced on a single squash fruit was quantified and estimated to be approximately three billion (Lamour et al. 2012). PATHOGEN HOST RANGE AND SYMPTOMS Phytophthora capsici was discovered in 1922 by Leon H. Leonian at the New Mexico Agricultural Research Station on chili pepper (Capsicum annuum L.) (Leonian 1922). Today, P. capsici is known to affect over 50 species, including economically important species in the Solanaceae, Fabaceae and Cucurbitaceae families and some Brassicaceae (Davidson et al. 2002, Hausbeck and Lamour, 2004, Gevens and Hausbeck 2005, Krasnow and Hausbeck 2015). Tian and Babadoost (2004) considered cucurbits and pepper to be most susceptible to P. capsici. The pathogen can cause root and crown rot, foliar and stem blight, seedling damping off, and fruit rot, depending on the host and environmental conditions (Zitter et al. 1996, Holmes et al. 2001, Islam et al. 2002, Babadoost 2000, 2004, 2005, Hausbeck and Lamour 2004). Generally, when P. capsici infects the roots of seedlings, damping off occurs, whereas wilting and plant death may occur in older plants (Lamour et al. 2012). In cucurbits, P. capsici can infect different parts of the plant including the root, crown, vines, leaves, and fruit. The disease often begins on plants located in the low areas of the field, where the water doesn’t drain, and the soil remains saturated for an extended period. Seedling damping off may occur pre- or post-emergence. Symptoms appear as watery lesions on the hypocotyls followed by wilting and death of the seedlings. Disease on cucurbit vines seems as watersoaked lesions that become dark olive to a brown color as the disease progresses. Disease symptoms on infected leaves are similar to those that appear on vines; lesions are initially watersoaked, become chlorotic, and then turn into necrotic lesions with chlorotic margins in a few days (Babadoost 2004). P. capsici may cause fruit rot, which begins with dark watersoaked tissue 5 followed by pathogen sporulation and then fruit degradation (Bababoost 2000, 2004, Meyer and Hausbeck 2013). Fruit infection often starts on the portion of the fruit that is in direct contact with the infested soil. However, rain splash or overhead irrigation may result in infested soil coming into contact with the upper fruit surface resulting in infection. Two to three days after fruit infection, abundant white sporangia have produced that look like powdered sugar on the fruit surface and result in damage to the whole fruit (Babadoost 2004, Gevens et al. 2007, Lamour et al. 2012, Meyer and Hausbeck 2013). Infected fruit tissue that remains in the field is a source of inoculum for new infection cycles during the growing season. The pathogen can form oospores when both mating types are present (Lamour and Hausbeck 2000, Hausbeck and Lamour 2004). Symptoms of P. capsici infection on less susceptible crops can include plant stunting, stem girdling, and cankers (Quesada and Hausbeck 2010). Susceptibility of different hosts or different cultivars of the same host is determined by the virulence and pathogenicity of the pathogen population in the field (Ristaino 1990, Kim and Hwang 1992, Foster and Hausbeck 2010a). Quesada et al. (2011) studied the population structure of P. capsici isolates to investigate the genetic diversity and pathogen virulence. They highlighted the importance of using isolates with diverse genetic backgrounds including isolates collected from temperate and tropical regions (Bowers et al. 2007) when screening cultivars for disease resistance in a breeding program. DISEASE MANAGEMENT STRATEGIES Disease management strategies include chemical and cultural practices (Ristaino and Johnston 1999, Babadoost 2004, Hausbeck and Lamour, 2004, Kousik et al. 2011). However, under conditions favorable for disease, significant yield losses still occur (Granke et al., 2012; Hausbeck 6 and Lamour, 2004). Several factors make P. capsici disease management challenging. The broad range of susceptible hosts and the persistence of P. capsici oospores in the soil limit the use of crop rotation (Kousik et al. 2015, Quesada et al. 2009, Lamour and Hausbeck 2003, Hausbeck and Lamour 2004). In fields infested with P. capsici, rotation with non-susceptible hosts for more than 5 years was not adequate to prevent crop loss of P. capsici susceptible crops (Lamour and Hausbeck 2003, Hausbeck and Lamour 2004). Cultural control methods include using well- drained fields, since water is a key factor in P. capsici dissemination and infection (Ristaino and Johnston 1999). Utilizing raised plant beds and trellises are recommended. However, in Michigan, winter squash and cucumbers grown for processing are mechanically harvested making it difficult to apply these cultural control methods. In areas with frequent rainfall such as the eastern U.S., cultural control methods are not effective when standing water is ≥ 2.5 cm in the field, creating suitable conditions for zoospore release and infection (Hausbeck and Lamour 2004). In combination with cultural methods and water management, fungicides can provide protection against P. capsici (Lamour and Hausbeck 2000, Hausbeck and Lamour 2004). The ability of the pathogen to reproduce sexually increases the likelihood of developing resistance to fungicides, such as cyazofamid and mefenoxam (Hausbeck and Lamour 2004, Jackson et al. 2012). Crops with fruits that have long maturation periods, such as winter squash and pumpkins, remain in direct contact with the infested soil for long periods of time increasing the likelihood of fruit rot (Granke et al. 2012). HOST RESISTANCE TO P. CAPSICI Host resistance is an essential element in strategies to control P. capsici. Commercial varieties and breeding lines of pepper have been screened for resistance to P. capsici (Barksdale et al. 1984, 7 Johnaston et al. 2002, Babadoost and Islam 2002, Oelke et al. 2003, Foster and Hausbeck 2010a). Foster and Hausbeck (2010a) also identified pepper lines and varieties with crown and root rot resistance to P. capsici isolates in Michigan. The fruit of these varieties was susceptible to P. capsici infection. The observed variation in susceptibility among pepper tissue types including root, stem, leaves, and fruit suggests that different mechanisms control resistance (Barksdale et al. 1984, Oelke et al. 2003). Complete resistance has not been detected in cucurbit commercial cultivars (Café-Filho et al. 1995). Many cucumber varieties have been screened for fruit resistance against fruit rot; however, only partial resistance has been identified (Gevens et al. 2006). In a greenhouse screen for crown rot resistance in a collection of commercial cultivars and germplasm accessions of summer squash, a green zucchini cultivar Spineless Beauty showed less susceptibility to crown rot than the other tested lines (Meyer and Hausbeck 2012). In a laboratory screen of different cucurbit fruits, yellow summer squash was more susceptible to fruit rot than winter squash, cucumber, watermelon, and melon (Ando et al. 2009). As complete resistance to P. capsici has not been identified in cucurbits, using available resistant cultivars in combination with the fungicides and cultural control may improve the control of P. capsici (Meyer and Hausbeck 2012). Variability in fruit rot susceptibility has been observed in different cucurbit fruits (Ando et al. 2009). Young fruits are more susceptible to fruit rot than older fruit (Lamour and Hausbeck 2004, Gevens et al. 2006, Ando et al. 2009, Meyer and Hausbeck 2013, Krasnow and Hausbeck 2016, Alzohairy et al. 2017). This phenomenon is known as age-related resistance (ARR), developmental resistance, or ontogenic resistance (Whalen 2005). Resistance is associated with specific developmental stages of the host (Stermer and Hammerschmidt 1984) and that resistance increases or decreases (Shah et al. 2015). Panter and Jones (2002), and Develey-Rivière and Galiana (2007) 8 described ARR in different plant-pathogen systems. Older hypocotyl tissue in the soybean seedling is resistant to P. sojae (Lazarovits et al., 1980). The young soybean hypocotyl tissue developed lesions when inoculated with P. sojae, while old tissue was resistant. ARR was correlated with the rapid production of the phytoalexin glyceollin in the older tissue (Ward et al. 1980); however, Lazarovits et al. (1981) showed that glyceollin was more correlated with necrosis than with resistance, suggesting other mechanisms control ARR. ARR to P. capsici has been described in some cucurbits and pepper (Ando et al. 2009, Kim et al. 1989, Biles et al. 1993). Pepper fruit acquires resistance to P. capsici as fruit ripening progresses (Biles et al. 1993). Cucumber fruit of a commercial cultivar and other genotypes of plant introductions exhibited ARR two weeks after fruit set. This ARR is likely correlated with the completion of fruit elongation period (Gevens et al. 2006). Other cucurbits exhibiting ARR at about 21 days post anthesis include Cucurbita moschata and Cucurbita pepo (Ando et al. 2009, Meyer and Hausbeck 2013, Krasnow and Hausbeck 2016). Some commercial cultivars of C. moschata develop resistance 14 days post anthesis (Alzohairy et al. 2017). The rates of fruit development are suggested to be responsible for determining the onset of ARR (Gevens et al. 2006). Therefore, cultivars/hosts with more days to maturity develop ARR later than those with fewer days to maturity. Selecting cultivars with ARR, especially those exhibiting ARR at earlier stages of fruit development could be beneficial to growers in the timing of fungicide spray and also reducing the number of applications (Ando et al. 2009, Hausbeck and Lamour 2004). The mechanisms behind ARR have been studied in several plants. Meyer and Hausbeck (2013) looked at the physiological and morphological changes as fruit age increased. Soluble solid contents and exocarp firmness were observed to increase with fruit maturing, but they were negatively correlated with the less susceptible phenotype of maturing fruit. Ando et al. (2009) 9 associated a change in fruit color and texture and increased waxiness with ARR in cultivars of C. pepo and C. moschata. Wounding negates ARR to P. capsici in cucumber (Granke and Hausbeck 2010), squash (Krasnow et al. 2014), and pepper (Biles et al. 1993), suggesting a role of the exocarp in resistance to P. capsici. In pepper, cuticle thickness increased with fruit maturity and increased resistance (Biles et al. 1993). Similarly, Ando et al. (2015) observed an increase in thickness of the cuticle and epidermal wall in cucumber, suggesting the role of a physical barrier. Generally, the plant cuticle is the first barrier that fungi and oomycetes have to penetrate in order to infect. The increase in cuticle thickness is a general protective mechanism against different pathogens such as detected in pepper against P. capsici (Biles et al. 1993). Also, at approximately three weeks old, bean hypocotyl cuticle thickness increases and becomes resistant to Rhizoctonia solani (Stockwell and Hanchey 1983). Plant production of secondary metabolites with antimicrobial activity is one of the primary defensive mechanisms against microbes (Dixon 2001, González-Lamothe et al. 2009). There are two classes of the defensive phytochemicals: low molecular weight preformed compounds that are produced and stored in plant tissues prior to pathogen infection, known as phytoanticipins, and low molecular weight induced compounds that are synthesized de novo after infection with pathogen, known as phytoalexins (VanEtten et al. 1994, Paxton 1981). The role of phytoanticipins and phytoalexins has been proven in several plant-pathogen systems. The phytoanticipins avenacin is produced by oat roots providing resistance against attack by the take-all disease of wheat caused by Gaeumannomyces graminis var. tritici (Osbourn et al. 1994). The role of avenacin in defense was demonstrated when a species of Avena, Avena longiglumis, was found to lack detectable amounts of avenacin and became infected with Gaeumannomyces graminis var. tritici. Also, oat plants are susceptible to G. graminis var. avenae that produces avenacinase, which detoxifies 10 avenacins (Osbourn et al. 1994). Also, the phytoanticipins dienes that are produced in unripe avocado fruit peel provide resistance against Colletotrichum gloeosporioides, the cause of anthracnose. Ripened fruit become susceptible to anthracnose as the dienes concentration decreases to levels that are not toxic to the pathogen (Prusky et al. 1991). A similar mechanism was found in mango fruit that possesses resorcinol in its unripen peel makes it resistant to Alternaria alternata (Droby et al. 1986, 1987), the levels of resorcinol in ripen fruit is nontoxic to A. alternata, and the fruit become susceptible to infection. The role of phytoalexins in defense has been described in several plant systems. Initially, phytoalexins were first described by Müller and Borger in 1940. Since then, more than 350 phytoalexins have been characterized in plant families, and 130 have been identified in the Leguminoseae family (Joseph 1995). The chemical structure of phytoalexins in the same family is mostly similar. For example, Solanaceae have terpenoid phytoalexins while Leguminoseae have oflavonoid phytoalexins (Singh and Chandrawat 2017). When phytoalexins are produced at the infection site, at the right time and antimicrobial concentrations, they are considered to be involved in defense (Hammerschmidt 1999, Hammerschmidt 2011). One of the best studied phytoalexins to be related to defense is camalexin. Camalexin was first isolated from leaves of Camelina sativa infected with Alternaria brassicae. It has been further identified in other crucifers (Glawischnig 2007). Camalexin is synthesized from tryptophan, and its production has been induced in Arabidopsis after infection by fungi, oomycetes, bacteria, and viruses (González-Lamothe et al. 2009). The role of camalexin as a defense mechanism in Arabidopsis has been proven. High concentrations of camalexin were detected at the infection site with A. alternata and locations near to lesions induced by Botrytis sp. (Kliebenstein et al. 2005). The accumulation of camalexin was correlated with induction of the precursor tryptophan and camalexin biosynthetic genes 11 (Schuhegger et al. 2007). Further, the role of camalexin in defense was proven in the mutant line pad3, which lacks the accumulation of camalexin. The pad3 mutant is susceptible to infection with Alternaria brassicicola and to B. cinerea isolates that were sensitive to camalexin (Kliebenstein et al. 2005). The phytoalexin C-glycosyl flavonoids, was found to be involved in resistance to powdery mildew in cucumber (McNally et al. 2003). When cucumber leaves were elicited with the plant-defense inducer Milsana before infection by the powdery mildew pathogen, the pathogen’s conidial chain growth collapsed within 48 hours after infection. Another phytoalexin, trans-p-coumaryl aldehyde, a lignin precursor, has been detected in the fruit of Cucurbita species including green acorn squash, butternut, and pumpkin (Stange et al. 1999). THE GENETIC BASIS OF RESISTANCE Early developing cucumber fruit are susceptible to P. capsici 8 days post pollination (dpp) and build resistance as the fruit age (Gevens et al. 2006). Transcriptomic studies of cucumber fruit across development identified distinct genes characterizing three phases of development. Fruits 0 – 4 dpp are characterized by a group of genes associated with cell division, organization, and biogenesis. Fruit aged 4 – 12 dpp are enriched in genes that function in cell structure and lipid metabolism. Fruit aged 12 – 16 dpp are enriched in genes associated with abiotic and biotic stress and stress-related transcription factors. The change in gene expression associated with fruit growth defense is correlated with the observed shift from young cucumber fruit susceptible to P. capsici to maturing fruits that are resistant (Ando et a. 2012). Transcript profiles of cucumber fruit peel of aging resistant fruit exhibit enrichment in genes associated with physical barriers, chemical defense, and molecular pattern-triggered or effector-triggered pathways (Ando et al. 2015). Another transcriptomic study of cucumber resistant fruit peel at 16 dpp revealed enrichment in genes encoding for flavonoid and terpenoid biosynthesis (Mansfeld et al. 2017). Further 12 metabolomic studies on the same resistant fruit ages showed enrichment in terpenoid glycosides more so than susceptible young cucumber fruit at 8 dpp, which suggests a correlation between production of terpenoid glycosides in resistant fruit and ARR to P. capsici (Mansfeld et al. 2017). ARR has been observed in apple leaves and fruit where disease susceptibility to Venturia inaequalis decreased with the aging of leaves and fruit. In a transcriptomic study of infected and non-infected leaves with Venturia inaequalis, five candidate genes were potentially correlated with ARR. One gene encodes for ‘enhanced disease susceptibility one protein’ was downregulated in both infected and non-infected leaves. The other four genes encode for metallothionein3-like protein, lipoxygenase, lipid transfer protein, and a peroxidase 3 were upregulated in both infected and non-infected leaves (Gusberti et al. 2013). These results suggested a potential mechanism of constitutive gene expression that is correlated with ARR in apple leaves. CONCLUSION Winter squash and pumpkin are economically important crops in the U.S. and worldwide. In Michigan and other U.S. states, fruit quality and yield are limited by P. capsici. Commercial cultivars are susceptible to fruit rot but ARR has been observed in commercial cultivars of C. moschata. Determining the onset of ARR in commercial cultivars and uncovering the mechanism of ARR in winter squash is helpful for integrated management strategies. 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Nature Publishing, John Wiley & Sons, Published on line 15.7.2003 (DOI: 10.1038/npg.els.0003723). Whalen, M. C. (2005). Host defence Pathology, 6:347-360. in a developmental context. Molecular Plant Whitaker, T. W., & Davis, G. N. (1962). Cucurbits. Botany, cultivation, and utilization. New York: Interscience Publisher. Inc. pp.250 Yoshikawa, M., Tsukadaira, T., Masago, H., & Minoura, S. (1977). A non-pectolytic protein from Phytophthora capsici that macertes plant tissue. Physiological Plant Pathology, 11:61-70. 21 Zitter, T. A., Hopkins, D. L., & Thomas, C. E. (1996). Compendium of Cucurbit Diseases (No. 635.62 C737). American Phytopathological Society, St. Paul, Minn. 22 CHAPTER I: CHANGES IN WINTER SQUASH FRUIT EXOCARP STRUCTURE ASSOCIATED WITH AGE RELATED RESISTANCE TO PHYTOPHTHORA CAPSICI ABSTRACT Phytophthora capsici is a destructive pathogen of cucurbits causing root, crown, and fruit rot. Winter squash (Cucurbita sp.) production is limited by this pathogen in Michigan and other U.S. growing regions. Age-related resistance (ARR) to P. capsici occurs in Cucurbita moschata fruit but is negated by wounding. The objective of this research was to determine if structural barriers exist in the intact exocarp of maturing fruit exhibiting ARR. Five winter squash (C. moschata) cultivars were evaluated for resistance to P. capsici 10, 14, 16, 18, and 21 days post pollination (dpp). ‘Chieftain’ butternut squash fruit expressing ARR 14 dpp were selected for analysis of exocarp cell wall changes and compared to 7 dpp (susceptible) and 21 dpp (resistant) using scanning electron microscopy (SEM). The cuticle, epidermal anticlinal walls, and cortex cell walls were measured and revealed significant increases in the cuticle and epidermal thicknesses as fruit age. P. capsici hyphae penetrated susceptible fruit 7 dpp directly from the surface or through wounds before 6- hour post inoculation (hpi) and completely degraded the fruit cell wall within 48 hpi. Resistant fruit, 14 dpp, and 21 dpp remained unaffected. Hyphae did not enter stomata 6 hpi. However, at 24 hpi the pathogen attempted to penetrate through stomata in the maturing resistant fruit. The high correlation between the formation of a thickened cuticle and epidermis in maturing winter squash fruit and resistance to P. capsici indicates the presence of a structural barrier to P. capsici as the fruit matures. 23 INTRODUCTION Phytophthora capsici Leonian, a destructive soilborne oomycete, infects vegetables including crops of cucurbits (Granke et al. 2012, Hausbeck and Lamour 2004, Kreutzer et al. 1940, Roberts et al. 2001, Tian and Babadoost 2004) and pepper (Foster and Hausbeck 2010), snap bean (Gevens and Hausbeck, 2005), and lima bean (Davidson et al., 2002). Under greenhouse conditions, P. capsici also infected Brassica spp. vegetable and biofumigation cover crops including cabbage, mustard, and radish (Krasnow and Hausbeck 2015). P. capsici limits cucurbit production in Michigan (Hausbeck and Lamour 2004, Meyer and Hausbeck 2012) and other U.S. production regions (Miller et al. 1994, Roberts et al. 2001, Babadoost 2004, Castro-Rocha et al. 2017). Nationally, Michigan ranks number one for production of summer and hard squash C. moschata and C. pepo repeatidly and fourth for C. maxima (pumpkin) production (USDA 2018). Due to the extended maturation period of 80-120 days for hard squash and pumpkin fruits, exposure to P. capsici-infested soil occurs for an extended time. Also, the dense plant canopy limits coverage of the fruit by fungicide sprays (Granke et al. 2012). Fruit rot can occur in the field (Granke et al. 2012) or postharvest (Hausbeck and Lamour 2004). Fruit rot signs and symptoms begin with dark water-soaked tissue followed by pathogen sporulation and fruit degradation (Badaboost 2004). Cucumber (Gevens et al. 2006, Ando et al. 2009, Hausbeck and Lamour 2004), butternut squash, melon, and watermelon (Ando et al. 2009), and pumpkin (Meyer and Hausbeck 2013) fruits are generally susceptible to P. capsici infection within 7 days post pollination. Integrated disease management strategies for P. capsici include raised plant beds with plastic mulch, water management, and fungicide application (Hausbeck and Lamour 2004, Ristaino and Johnston 1999). Raised plant beds are not suitable for crops that will be mechanically harvested 24 such as hard-rind squash and pumpkins used for processing (Granke et al. 2012). However, the use of plant beds with plastic mulch was successful in increasing yield and improving quality in many vegetable crops such as cucumbers, tomatoes, pepper, and eggplant (Lamont 2017). Crop rotation offers limited relief since the pathogen produces thick-walled oospores that can survive in the soil for many years in the absence of a host (Babadoost 2005, 2013). P. capsici produces zoosporangia and swimming zoospores that are disseminated by rainwater splash or irrigation water (Bowers et al. 1990, Granke et al. 2012, Babadoost 2004). The pathogen undergoes sexual recombination, increasing the likelihood of developing resistance to fungicides which has occurred for mefenoxam and metalaxyl (Lamour and Hausbeck 2004) and cyazofamid (Kousik and Keinath 2008). Host resistance along with other disease management strategies can provide long-term management strategy against P. capsici (Hausbeck and Lamour 2004, Quesada and Hausbeck 2010, Granke et al. 2012). Although complete resistance has not been identified in commercial cultivars of cucurbits (Café-Filho et al. 1995), age-related resistance (ARR) has been observed in different cucurbits (Gevens et al. 2006, Ando et al. 2009, Krasnow et al. 2016, Meyer and Hausbeck 2013). ARR or ontogenic resistance is resistance associated with specific host developmental stages (Stermer and Hammerschmidt 1984, Hammerschmidt 2015). ARR has been observed in different host-pathogen interactions (Lazarovits et al. 1981, Kim et al. 1989, Kus et al. 2002, Panter et al. 2002). Cucurbits and Solanaceae vegetable crops express ARR to P. capsici (Hausbeck and Lamour 2004, Gevens et al. 2006, Ando et al. 2009, Krasnow et al. 2016, Meyer and Hausbeck 2013, Biles et al. 1993). Squash (Cucurbita pepo, Cucurbita moschata) (Ando et al. 2009, Krasnow et al. 2016), cucumber (Hausbeck and Lamour 2004, Gevens et al. 2006, Ando 25 et al. 2009), and pumpkin (C. moschata) (Krasnow et al. 2016, Meyer and Hausbeck 2013) develop resistance to fruit rot as they mature. The time of ARR is variable among cucurbits (Ando et al. 2009, Meyer and Hausbeck 2013). It has been observed in cucumber that when the fruit reached its maximum length that overlapped with the resistance against P. capsici (Gevens et al. 2006, Ando et al. 2009, Ando et al. 2015). Complete fruit elongation can vary in different cucurbits, wherein cucurbits are producing large fruit such as pumpkins and squash, the fruit size reaches its maximum between 20-24 days-post pollination (dpp). For cucurbits producing small fruit, the fruit maximum size is achieved between 15-20 dpp (Loy 2004). In cucumber, fruit full length is achieved 10-12 dpp and corresponds to fruit resistance (Ando et al. 2009). Processing pumpkins C. maxima ‘Golden Delicious’ and C. moschata ‘Dickenson Field’ have a relatively long maturation period. ‘Dickenson Field’ develops resistance to P. capsici 21 dpp whereas ‘Golden Delicious’ remains susceptible (Meyer and Hausbeck 2013). The mechanisms controlling ARR to P. capsici has been studied in different cucurbits (Mansfeld et al. 2017, Meyer and Hausbeck 2013, Krasnow and Hausbeck 2016) and pepper (Biles et al. 1993). Wounding resistant fruit negated ARR (Krasnow et al. 2014, Biles et al. 1993) suggesting the fruit exocarp provides resistance to maturing fruit. The objectives of this study included determining: 1) the onset of ARR to P. capsici during fruit development among five C. moschata commercial cultivars and 2) the structural changes in the fruit exocarp cell wall and P. capsici hyphal penetration mechanisms in young susceptible and maturing resistant fruit using scanning electron microscopy. 26 MATERIALS AND METHODS Five butternut winter squash and processing pumpkin cultivars of Cucurbita moschata were selected (Table 1.1). Seeds were planted on 15 June 2015 into 72-cell trays containing soilless peat mixture (Suremix Michigan Grower Products, Inc. Galesburg, MI) and grown for two weeks in the research greenhouse at Michigan State University (MSU) in East Lansing, MI. Thirty seedlings from each cultivar were transplanted on 1 July to a field site, previously planted to pumpkin, at the MSU Plant Pathology Farm in Lansing, MI. The soil type was Capac loam with no known P. capsici infestation. Plants were grown on raised plant beds covered with plastic mulch and irrigated twice each week via drip emitters. Plant rows were 30.5 m long with 3.7 m between rows and 61 cm between plants. At anthesis, female flowers were hand-pollinated and tagged with the date. Fruits were harvested 10, 14, 16, 18, and 21 days post pollination (dpp) similar to Meyer and Hausbeck (2013) with modification. 27 Table 1.1.: Cucurbita moschata with listed cultivars used in the study. Cultivar Chieftain Waltham Early Avalon Dickenson Field Cultivar type Butternut squash Butternut squash Butternut squash Butternut squash Processing pumpkin Days to maturity 80 110 82 90 100 Source Rupp Seeds Inc., Wauseon, OH Rupp Seeds Inc., Wauseon, OH Rupp Seeds Inc., Wauseon, OH Rupp Seeds Inc., Wauseon, OH Rispens seeds Inc. Phytophthora capsici isolate 12889 (mating type A1, insensitive to mefenoxam) from bell pepper (Foster and Hausbeck 2010) was selected from the long-term collection of M.K. Hausbeck at MSU. To confirm pathogen virulence prior to inoculation, the isolate was used to inoculate cucumber fruit, then recovered from the infected fruit, and maintained on unclarified V8 agar (143 mL V8 juice, 3g CaCO3, 16 g agar, 850 mL distilled water) (Dhingra and Sinclair 1985, Krasnow et al. 2017) under constant fluorescent light at room temperature (21 ± 2oC). Before inoculation, fruits were surface disinfested with 0.4% of sodium hypochlorite solution for 5 min, rinsed with water for 2 min, and allowed to air dry. A 7-mm V8 agar plug, removed from an actively growing 7 to 9-day old P. capsici culture using a cork borer, was used to inoculate the fruit. The agar plug was placed mycelial side down on the fruit at the mid-point between the peduncle and blossom end and covered with a sterilized screw cap (16.5 mm in diameter) (Axygen Inc., Union City, CA) that was fixed to the fruit with petroleum jelly. A sterile, uncolonized agar plug was used for control fruit. Fruits were incubated in 99 L or 62 L clear plastic bins (Sterilite) lined on the inside edges with water-saturated paper towels to maintain high relative humidity and kept at room temperature (22±2°C) under constant fluorescent light (Meyer and Hausbeck et al. 2012, Krasnow et al. 2014). 28 Disease severity was assessed four days post inoculation (dpi) by measuring the diameter of the lesion and pathogen growth. A rating scale from 0 to 4 was used to visually assess the pathogen growth density, where 0 = no visible pathogen growth; 1 = water-soaked tissue only; 2 = light visible mycelial growth; 3 = moderate mycelial growth; and 4 = dense mycelial growth (Meyer and Hausbeck et al. 2013, Krasnow et al. 2014, Krasnow and Hausbeck 2016). According to Krasnow and Hausbeck (2016), fruits were considered resistant with a mean disease rating of <0.5 and intermediately resistant with a mean disease rating >0.5 and <1.5. Disease incidence was calculated as a percentage of infected fruits (Krasnow and Hausbeck 2016). After the assessment, a small (1-2 mm) tissue segment at the leading edge of symptomatic tissue was removed and placed onto V8 agar with ampicillin, rifampicin, pentachloronitrobenzene (PCNB) and benomyl. The recovered isolate was confirmed as P. capsici using morphological characteristics (Waterhouse 1963). ‘Chieftain’ butternut squash fruits were selected for scanning electron microscopy (SEM) studies. In mid-May 2016, seeds were planted and grown as previously described. Fruits were harvested 7, 14, and 21 dpp and disinfested as previously described. Nine nonwounded fruit of 7, 14, and 21 dpp were inoculated with P. capsici at three sites as technical replicates as described previously. At 6, 24, and 48 h post inoculation (hpi), cross sections (~2.0 mm x 6-10.0 mm x 2.0 mm; thickness/width/depth) from the three inoculated sites of three fruit/hpi were prepared at the Center for Advanced Microscopy at MSU. Briefly, samples were fixed at 4oC for a minimum of 1 h in 4% glutaraldehyde buffered with 0.1 m sodium phosphate at pH 7.4, then rinsed briefly in the buffer and dehydrated in an ethanol series (25%, 50%, 75%, 95%, 3X 100%) for 1 h. Samples were freeze-dried in an Electron Microscopy Sciences Model EMS750X freeze dryer (Electron Microscopy Sciences, Hatfield, PA), and then mounted onto aluminum stubs using adhesive tabs 29 (M.E. Taylor Engineering, Brookville, MD). Samples were coated with osmium (~10.0 nm thickness) in a NEOC-AT osmium coater (Meiwafosis CO., Ltd., Osaka, Japan). Images for the cross-sections and top surface of each sample’s inoculation sites were examined using SEM. These fruits were also used to exam exocarp structural differences at non-wounded and non- inoculated sites. Cross sections (~2.0 mm x 6-10.0 mm x 2.0 mm; thickness/width/depth) of non- wounded and non-inoculated sites of nine fruit/age were prepared as described above. Transverse sections were prepared from nine fruit/age to examine the exocarp surface. After the transverse sections were removed from the fruit, the samples were immediately frozen in liquid N2, placed in an aluminum freeze drier basket, and allowed to slowly warm to room temperature on aluminum- stubs in a desiccator. Both cross and transverse sections were examined under the 6610LV SEM (JEOL Ltd. Tokyo, Japan) and images recorded using software version 3.08 of SEM Control User Interface (JEOL Technics Ltd., Tokyo, Japan). Image J software, a public domain Java image-processing program (Jensen 1986), was used to visualize the cross-sections images and measure the thickness of different cell wall structures including the cuticle, the top- and mid-point of the epidermal anticlinal walls, and the cortex cell walls of the first four layers below the epidermis. ANOVA (P = 0.05) was used to detect significant differences among 7, 14, and 21 dpp fruit ages at the cuticle, the top- and mid-point epidermal anticlinal walls and the cortex cell wall. When a significant difference was found in cell wall structures among ages, pairwise comparisons were achieved using Tukey’s Honest Significant Difference (HSD) test (P = 0.05) to determine which age(s) were different. All statistical analyses were conducted using R statistical software (R Development Core Team, 2014). 30 RESULTS Fruits of all cultivars harvested 10 dpp developed disease symptoms four days post inoculation and disease incidence across cultivars ranged from 44.4% to 100% (Table 1.2). The onset of ARR was variable among cultivars (Table 1.2, Figure 1.1). ‘Early’ and ‘Chieftain’ butternut squash showed resistance (0% disease) beginning 14 dpp and remained resistant at 16, 18, and 21 dpp. ‘Dickenson Field’ fruit exhibited ARR 21 dpp with 11% disease. ‘Avalon’ fruit were resistant 14 dpp but not at 16 dpp. ‘Avalon’ fruit 18 and 21 dpp were resistant (0% disease). ‘Waltham’ butternut fruit had 11% disease, 18 and 21 dpp. Average disease severity ratings from 10 to 21 dpp for cultivars Early, Chieftain, and Dickenson Field ranged from 0.8 to 0, 2.4 to 0, and 3.2 to 0.1, respectively (Table 1.2, Figure 1.1). For ‘Waltham’ and ‘Avalon’, the highest (1.2 and 1.4) and lowest (0.1 and 0) disease ratings occurred 16 and 18 dpp, respectively (Table 1.2, Figure 1.1). Lesion diameter (LD) decreased as fruit aged in ‘Early’, ‘Chieftain’, and ‘Dickenson Field’, while LD in ‘Waltham’ and ‘Avalon’ followed the same pattern as their disease ratings (Table 1.2, Figure 1.1), except that Avalon was not diseased at 14 dpp. 31 Table 1.2.: Disease rating, lesion diameter and disease incidence four days post inoculation with P. capsici for 5 cultivars of Cucurbita moschata at fruit developmental stages 10, 14, 16, 18, and 21 days post pollination (dpp). Disease rating (0-4 scale)a 0.8 2.4 3.2 0.8 1 0 0 2.3 0.4 0 0 0 1.6 1.2 1.4 0 0 0.7 0.1 0 0 0 0.1 0.2 0 Fruit age (dpp) Cultivar 10 dpp Early Chieftain Dickenson Field Waltham Avalon 14 dpp Early Chieftain Dickenson Field Waltham Avalon 16 dpp Early Chieftain Dickenson Field Waltham Avalon 18 dpp Early Chieftain Dickenson Field Waltham Avalon 21 dpp Early Chieftain Dickenson Field Waltham Avalon a, b Disease rating and lesion diameter values represent mean of 9 fruits conducted in three experimental replicates per age and cultivar. Disease scale was 0-4 where 0 is no visible infection, 1 is water soaking, 2 is light pathogen mycelial growth, 3 is moderate pathogen mycelial growth and 4 is extensive pathogen mycelial growth. Rating was done 4 days post inoculation (dpi). Disease incidence (%) 44 78 100 44 44 0 0 78 33 0 0 0 56 33 44 0 0 44 11 0 0 0 11 11 0 Lesion diameter (cm)b 0.7 3.2 6.2 1.7 1.9 0 0 5.2 1.1 0 0 0 3.2 2.5 2.3 0 0 2.4 0.6 0 0 0 0.5 0.7 0 32 g n i t a r e s a e s D i 4 3 2 1 0 Early Dickenson Field Chieftain Waltham Avalon 10 14 18 Days post pollination 16 21 Figure 1.1.: Average disease rating of Cucurbita moschata cultivars’ fruits at 10, 14, 16, 18, and 21 dpp in response to Phytophthora capsici inoculation, four days post inoculation (disease rating 0 = no visible pathogen growth; 1 = water-soaked tissue only; 2 = light visible mycelial growth; 3 = moderate mycelial growth; and 4 = dense mycelial growth). SEM images revealed increases in the thickness of the cuticle and epidermal anticlinal walls as fruit aged from 7 to 21 dpp (Figure 1.2). The thickness of cuticle and epidermal cell walls increased as the fruit aged but not the cortex cell walls (Table 1.3). Significant differences in the thickness of the cell wall structure among ages were detected (ANOVA P £ 0.05). Pairwise comparisons between ages indicated significant differences at the cuticle and both points of epidermal anticlinal walls between 7 and 14 dpp, and 7 and 21 dpp (Figure 1.3, Table 1.4). Significant differences between 14 and 21 dpp were detected in the cuticle and top-point of epidermal anticlinal walls thicknesses; differences were not detected in thickness of the mid-point of the epidermal anticlinal walls. Cortex cell wall thickness measurements were not significantly different between 7 and 14 dpp (P = 0.9), while differences between means of 7 and 21 dpp (P = 0.001), and 14 and 21 dpp 33 (P = 0.0004) were significant. SEM images of transverse sections of 7, 14, and 21 dpp showed an increase in the wax deposition on the fruit surface as the fruit aged (Figure 1.4). Figure 1.2.: Scanning electron microscopy images of cross sections of non-wounded/non- inoculated fruits of cultivar Chieftain (a) 7 dpp, (b) 14 dpp, and (c) 21 dpp. Arrows for (c) represent the cell wall structures and the measurement direction where a: cuticle, b: epidermal anticlinal wall top-point, c: epidermal anticlinal wall mid-point, and d: cortex cell wall. Arrows heads represent the direction of measurement. 34 Table 1.3.: Minimum and maximum thickness (µm) of cell wall structures: cuticle, epidermal anticlinal wall top-point, epidermal anticlinal wall mid-point, and cortex cell wall across fruit development at 7, 14, and 21 days post pollination (dpp) in cultivar Chieftain. Days post pollination (dpp) Cell wall structures Thickness (µm) (Min-Max) 7 14 21 Cuticle Epidermal anticlinal wall top-point Epidermal anticlinal wall mid-point Cortex Cuticle Epidermal anticlinal wall top- point Epidermal anticlinal wall mid- point Cortex Cuticle Epidermal anticlinal wall top- point Epidermal anticlinal wall mid- point Cortex 0.5-0.9 1.9-2.5 0.9-1.7 0.7-0.9 1.1-1.5 3.8-4.6 2.7-4.7 0.7-0.8 1.3-1.7 5.0-7.8 3.6-4.9 0.8-1.0 Table 1.4.: Pairwise comparisons of cell wall structures between fruit developmental stages of cultivar Chieftain. Days post pollination (dpp) Cuticle 7 vs 14 7 vs 21 14 vs 21 <0.000001* <0.000001* 0.02* 2e-07* *Indicates significant differences between ages in the rows (P £ 0.05), HSD 0.05 (Tukey’s Honest Significant Difference test) Epidermal anticlinal wall top-point 5e-07* <0.0000005* Epidermal anticlinal wall mid-point <0.00000005* <0.00000001* 0.13 Cortex 0.9 0.001* 0.0004* 35 Figure 1.3.: Histogram showing average thickness (µm) of cell wall structures: cuticle, epidermal anticlinal wall top-point, epidermal anticlinal wall mid-point, and cortex cell wall across fruit development at 7, 14, and 21 dpp of cultivar Chieftain. Same color column with different letter indicates significant difference (P £ 0.05) of cell wall structure thickness across ages. Figure 1.4.: Scanning electron microscopy images of transverse sections at non-wounded/non- inoculated fruits of cultivar Chieftain. (a) 7 dpp, (b) 14 dpp, and (c) 21 dpp. Wax appears as spiny crystals on fruit surface. When cross sections of 7, 14, and 21 dpp were examined 6 and 24 hpi the cell wall did not appear affected by P. capsici (Figure 1.5a-e). While complete cell wall collapse and fruit tissue degradation were detected 48 hpi for 7 dpp fruits, the tissue of fruit harvested 14 and 21 dpp remained unaffected (Figure 1.5g-i). Cross sections of 7 dpp fruit showed that hyphae penetrated 36 the epidermal layer before 6 hpi and was detected around the vascular bundles at 48 hpi (Figure 1.6a, b). Cross section images at 14 and 21 dpp detected no signs of hyphal penetration of the fruit tissue among the three-time intervals (Figure 1.6c-h). Figure 1.5.: SEM images of cross sections at 6 hour post inoculation (hpi), 24 hpi, and 48 hpi of cultivar Chieftain fruits at (a, d, g) 7 dpp, (b, e, h) 14 dpp, and (c, f, i) 21 dpp, respectively, showing the effect of Phytophthora capsici inoculation on cell wall integrity. The white circles point for the inoculation site. 37 Figure 1.6.: Scanning electron microscopy images of cross sections of cultivar Chieftain fruits of 7 days post pollination (dpp) at (a) 6 hours post inoculation (hpi), (b) 48 hpi; 14 dpp at (c) 6 hpi, (d) 24 hpi, (e) 48 hpi; and 21 dpp at (f) 6 hpi, (g) 24 hpi, and (h) 48 hpi. Hy=hypha; c=cuticle; eaw=epidermal anticlinal wall; vb=vascular bundle. Top surface views 6 hpi of the same cross-section samples from fruit harvested 7 dpp revealed hyphae directly penetrating the epidermal surface (Figure 1.7a). Hyphae were observed growing over stomata without entering, bypassing multiple stoma cells without penetrating and growing towards a wound (Figure 1.7b, c). At 24 hpi, hyphae were branched and penetrated the epidermal surface directly, and by 48 hpi multiple hyphal penetration points were detected in 7 dpp fruits (Figure 1.7d, e). Top surface images of 14 and 21 dpp detected no direct hyphal penetration across 38 the three-time intervals, and hyphae did not enter through stomata (Figure 1.7f, g). However, hyphae were observed penetrating stomata at 24 hpi in both 14 and 21 dpp fruits (Figure 1.7 h, i). At 48 hpi, one appressorium was detected suggesting a direct penetration attempt in 14 dpp fruits (Figure 1.7j). However, visible hyphal penetration was not detected in 21 dpp fruits at 48 hpi. 39 Figure 1.7.: Scanning electron microscopy images of the top surface view of cultivar Chieftain fruits. (a) 7 days post pollination (dpp) at 6 hours post inoculation (hpi), hyphal direct penetration from the epidermal surface (b) 7 dpp at 6 hpi, hypha passing over stomata without entering, (c) 7 dpp at 6 hpi, hypha traveling toward entering through wound and bypassing multiple stomata; (d) 7 dpp at 24 hpi, hypha branching and directly penetrating; (e) 7 dpp at 48 hpi, multiple penetration points; (f, g) 14 dpp and 21 dpp, respectively, at 6 hpi showing hyphae passing over stomata but not entering; (h, i) 14 dpp and 21 dpp, respectively, at 24 hpi showing hyphae entering through stomata; sr=surface ridge; es=epidermal surface; hy=hypha; st=stomata; w=wound; ap= appressorium. 40 DISCUSSION In this study, we aimed to study the mechanism of ARR to P. capsici in C. moschcata cultivars through determining the onset of resistance, studying the fruit exocarp structural changes across development and uncovering the mechanism of hyphal penetration of winter squash fruit. Once a production site becomes infested with P. capsici, growers are encouraged to utilize all available tools to manage the disease. Understanding the mechanism of ARR in winter squash is beneficial to growers where choosing a cultivar known to develop ARR can be used as part of an integrated strategy that combines other cultural strategies and effective fungicides. ARR onset differed among the five cultivars of C. moschata included in this study. The onset of ARR in ‘Dickenson Field’, a processing pumpkin, occurred 21 dpp with <11% diseased fruit, consistent with previous findings (Meyer and Hausbeck 2013). This cultivar was used as a control for our study and included fruit aged 16 and 18 dpp that were not tested previously. Results showed a gradual decrease in susceptibility to P. capsici as the fruit aged, suggesting changes in fruit development are associated with pathogen resistance. Krasnow and Hausbeck (2016) evaluated several butternut squash cultivars including ‘Waltham’, and ‘Avalon’ at 7, 14, 22, and 56 dpp and found them to be intermediately resistant to P. capsici at 14 dpp and completely resistant 22 dpp. Our results found that at 16 dpp ‘Waltham’ and ‘Avalon’ were intermediately resistant and became resistant at 21 and 18 dpp, respectively. ‘Early’ and ‘Chieftain’ became completely resistant at 14 dpp and older. Variable days to maturity among cultivars might be the cause for the observable difference in the onset of ARR. ‘Dickenson Field’, ‘Waltham’, and ‘Avalon’ require more days to mature than ‘Early’ and ‘Chieftain’, which may suggest that ‘Early’ and ‘Chieftain’ reach full fruit elongation sooner than other cultivars. Fruit transition from a susceptible to resistant state is associated with complete fruit elongation stage (Gevens et al. 2006, Ando et al. 2009, Ando et al. 2015, Krasnow 41 and Hausbeck 2016) and maybe the reason we detected an earlier onset of ARR at 14 dpp in ‘Chieftain’ and ‘Early’ than in other cultivars. The underlying mechanisms of ARR have been investigated in other plant systems including cucumber (Ando et al. 2015) and pepper (Biles et al. 1993); however, different plant species and even different cultivars of the same species can have different mechanisms (Panter & Jones, 2002; Whalen, 2005, Develey-Rivière and Galiana 2007). Structural changes are important factor in plant defense against insects and pathogens (Freeman and Beattie 2008, War et al. 2012). The fruit cell wall develops a physical barrier against pathogen attack and provides the first layer of protection (Freeman and Beattie 2008). In the SEM study of ‘Chieftain’ fruit, cell wall collapse and degradation in 7 dpp fruit were observed within 48 hpi from P. capsici, while older (14 and 21 dpp) fruit remained unaffected (Figure 1.5), this suggests a physical barrier prevents hyphal penetration by the pathogen to the inner layer of fruit cell wall. In plants, epicuticular wax deposition increases as plant age increase (Maiti 2012). Epicuticular wax is a hydrophobic layer which reduces the wet environment needed for pathogen spores to attach and germinate (Maiti 2012). In our study, wax deposition increased with aging fruit that demonstrated resistance to P. capsici (Figure 1.4), suggesting the formation of a physical barrier for pathogen hyphal penetration. Wounding negates ARR (Krasnow et al. 2014, Biles et al. 1993), thus supporting the hypothesis that intact fruit exocarp is essential for resistance. This study and previous studies found nonwounded fruits of different cucurbits exhibit ARR to P. capsici as they age (Ando et al. 2009, Gevens et al. 2006, Meyer and Hausbeck 2013, Krasnow and Hausbeck 2016). However, a study with pepper demonstrated resistance to P. capsici associated with fruit maturity and the ripening process as the fruit changes from green to red (Biles et al. 1993). An increase in cuticle thickness with fruit maturity was detected in pepper, suggesting a role of the 42 cuticle as a physical barrier to P. capsici infection (Biles et al. 1993). A similar phenotype was detected in cucumber fruit; cuticle and epidermal cell wall thickness were increased in resistant fruit 16 dpp when compared to susceptible fruit 8 dpp (Ando et al. 2015). The resistant phenotype of tomato fruit at the stem end to P. capsici is correlated with its thick cuticle and epidermal cell walls (Simonds and Kreutzer 1944). ARR and the role of cuticle thickness in defense are not unique to P. capsici. In bean, the hypocotyl becomes resistant with age to Rhizoctonia solani as its cuticle thickness increases (Stockwell and Hanchey 1983). Peach genotypes resistant to Monilinia fructicola have thicker cuticles than the susceptible genotypes (Adaskaveg et al. 1989). When the anatomy of C. maxima buttercup squash fruit across developmental stages was studied, the cuticle and cell wall thickness increase was observed with maturity (Sutherland and Hallett 1993). Our study showed an increase in the cuticle and epidermal anticlinal wall thickness (Figure 1.2) and wax deposition (Figure 1.4) in aging resistant ‘Chieftain’ fruit compared to a reduced thickness of the same structures and less wax deposition detected in young, susceptible fruit. Our findings suggest that the cuticle and epidermis act as a physical barrier to pathogen penetration and provide resistance to maturing fruit. A lower level of significance was detected between the thickness of cortex cell walls of young (7 dpp) susceptible and older (21 dpp) resistant fruit compared to a higher level of significance detected in cuticle and epidermis (Figure 1.3). Thus, the cortex may play a less critical role in resistance since the difference in thickness of cortex cell walls between 7 dpp susceptible fruit and 14 dpp resistant fruit were not significant. Phytophthora capsici penetrates the plant surface directly or through natural openings such as stomata (Hausbeck and Lamour 2004). On pepper leaves, Du et al. (2013) noted that P. capsici zoospores encysted within three hours and then produced two germ tubes that penetrated the surface directly; an appressorium was not detected. In our study, P. capsici hyphae penetrated 43 susceptible fruit tissue before 6 hpi (Figure 1.6a). Appressorium formation was detected 24 hpi on the exocarp surface of the resistant fruit (Figure 1.7j), suggesting an attempt by the pathogen to penetrate the tissue using mechanical force. When fruit were harvested 7 dpp, pathogen hyphae penetrated directly or through wounds by 6 hpi (Figure 1.7a, c). Hyphae were embedded in the epidermal cells of young susceptible fruit (Figure 1.6a). We did not observe penetration through stomata of any of the squash fruit regardless of age 6 hpi (Figure 7b, f). Hyphae were observed growing over the stomata without penetrating (Figure 7b, f), similar to what Du et al. (2013) observed in pepper leaves inoculated with P. capsici. This suggests that penetration through stomata is not preferred by the pathogen. A similar pattern was observed with Cercospora henningsii where the pathogen passed over the stomata without entering (Babu et al. 2009). However, a 24 hpi, we detected hyphal penetration through the stomata in squash fruit harvested 14 and 21 dpp was detected (Figure 7h, i), which suggests that when the pathogen encounters obstacles preventing its direct penetration through the surface, it attempts to penetrate through the stomata. Barriers to hyphal penetration might also be present in cells in the substomatal cavity since hyphae were not detected inside the resistant fruit tissue (Figure 1.6c- h). Krasnow and Hausbeck (2016) found that P. capsici zoospores accumulated over stomata of a susceptible C. maxima cultivar while no accumulation was detected on a C. moschata cultivar, which exhibits ARR. Mycelia colonized the vascular bundles of the susceptible 7 dpp fruit (Figure 1.6b), while colonization was not observed at or near the vascular bundles in the 14 and 21 dpp resistant fruit. Similar results were reported with Phytophthora sojae on soybean where the pathogen colonized the vascular tissue of the roots of a susceptible cultivar but not of a resistant cultivar (Enkerli et al. 1997). When fruit were harvested 7 dpp, P. capsici colonized the cuticle, epidermis, cortex, and 44 vascular tissue within 48 hpi (Figure 1.6b). Colonization was not detected in fruit harvested 14 and 21 dpp (Figure 1.6c -h). Fruit harvested 14 dpp or later, exhibited ARR (Figure 1.1). Our results are consistent with a study by Kim and Kim (2009) where P. capsici colonized the epidermis, cortex and vascular tissue of pepper roots in a susceptible cultivar but did not colonize the vascular tissue of a resistant cultivar. The pathogen was not able to infect or colonize the roots of the resistant cultivar roots due to root structural defenses (Kim and Kim 2009). In conclusion, our study showed a high correlation between the thickness of cell wall structures including cuticle and epidermis, and ARR (Figure 1.3, Table 1.4)), suggesting the presence of a structural barrier to P. capsici invasion of maturing fruit tissue. Selection of germplasm with resistance to P. capsici is of interest to plant breeders and growers, however, complete plant resistance has not been identified. ARR could be a valuable phenotype to integrate into breeding programs. The incorporation of cultivars expressing ARR early (i.e. 14 dpp) could benefit management of P. capsici. Since the early onset of ARR might be related to fewer days to maturity, growers could select cultivars with desired horticultural characteristics that express ARR with fewer days to maturity. 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Molecular Plant 50 CHAPTER II: ANTIFUNGAL ACTIVITY IN WINTER SQUASH FRUIT PEEL IN RELATION TO AGE RELATED RESISTANCE TO PHYTOPHTHORA CAPSICI ABSTRACT Age-related resistance (ARR), or ontogenic resistance, is associated with host developmental stages. Winter squash fruit (Cucurbita moschata) develops resistance to the oomycete plant pathogen Phytophthora capsici as they mature. P. capsici is soilborne and infects cucurbits, solanaceous crops, lima and snap beans causing damping off, foliar blight, and root, crown rot, and fruit rot. In Michigan and other U.S. growing regions, winter squash production is limited by P. capsici. Infection of cucurbit fruit by P. capsici appears first as a water-soaked lesion, followed by pathogen sporulation, and fruit rot. ARR in winter squash to P. capsici could be exploited to assist growers in limiting crop loss. Previous research suggested the presence of a preformed structural barrier to P. capsici in resistant mature fruit exocarp. However, antifungal compounds in the fruit exocarp might also be associated with ARR. The objective of this study to determine whether preformed or induced antifungal activity during fruit development is correlated with ARR. Three winter squash cultivars demonstrating ARR were hand-pollinated and then harvested 10, 14 and 21 days post pollination (dpp). Fruits were peeled with three replicates/dpp/cultivar, and a methanol extract was prepared. A thin-layer chromatography (TLC) bioassay using Cladosporium cucumerinum was used to detect antifungal activity in winter squash peel extracts. Results indicated the presence of compounds with antifungal activity in all fruit ages tested, but the antifungal activity decreased with age indicating a lack of correlation between preformed antifungal activity and ARR in winter squash. Induced antifungal activity in winter squash fruit peel from fruits 7, 10, 14 and 21 dpp was examined following inoculation with a P. capsici mycelial agar plug at non-wounded sites. Results indicated no significant change in the 51 antifungal activity among fruit ages regardless of the post inoculation time intervals suggesting no correlation between the induced antifungal activity and ARR in winter squash. INTRODUCTION Fruit rot caused by Phytophthora capsici is a limiting problem for squash producers in Michigan (Hausbeck and Lamour 2004, Lamour et al. 2012, Granke et al. 2012) and other states (Miller et al. 1994, Ristaino 1999, Robert et al. 2005) with entire fields of squash becoming infected (Meyer and Hausbeck 2012). Michigan is ranked number one for summer and hard squash (USDA 2018) where the crops are used for fresh market and processing. Disease symptoms include root and crown rot, fruit rot, and foliar blight (Babadoost 2005). Long-lived oospores in the soil, an expansive host range, and the development of pathogen resistance to fungicide challenges current management strategies (Hausbeck and Lamour 2004, Quesada-Ocampo et al. 2009, Lamour and Hausbeck 2000). Commercial cultivars with host resistance are not currently available. Age-related (ARR) or ontogenic resistance is the development of pathogen resistance during developmental stages of the plant and/or fruit (Stermer and Hammerschmidt, 1984; Hammerschmidt 2015). ARR has been observed in several plant-pathogen systems including Arabidopsis and Pseudomonas syringae pv. tomato, Apple leaves and Venturia inaequalis, and soybean seedlings and Phytophthora sojae (Panter and Jones 2002, Develey-Rivière et al. 2007). ARR to P. capsici has been demonstrated with fruits of pepper (Biles et al. 1993), cucumber (Ando et al. 2009, Gevens et al. 2006), and squash (Ando et al. 2009, Meyer and Hausbeck 2013, Krasnow et al. 2014, Krasnow and Hausbeck 2016, Alzohairy et al. 2017). While the onset of ARR is variable among species and cultivars, the early developmental stage of all cucurbit fruits is susceptible to P. capsici (Gevens et al. 2006, Ando et al. 2009, Meyer and Hausbeck 2013, 52 Alzohairy et al. 2017). When resistant squash fruit 21 to 24 days post pollination (dpp) were wounded, they became susceptible to P. capsici (Krasnow et al. 2014) suggesting that the fruit exocarp forms a structural or chemical barrier to P. capsici. Plants produce secondary metabolites with antimicrobial activity as a chemical defense mechanism against pathogens (Dixon 2001, González-Lamothe et al. 2009). Phytoanticipins are low molecular weight antimicrobial phytochemical compounds produced from preexisting constituents that play a role in plant defense (VanEtten et al. 1994, González-Lamothe et al. 2009, Singh and Chandrawat 2017) and include saponins, glucosinolates, and cyanogenic glycosides (Piasecka et al. 2015). Avencains are a saponin produced by oat roots that serve as a chemical barrier against the take-all disease of wheat caused by Gaeumannomyces graminis var. tritici (Osbourn et al. 1994). Phytoalexins are low molecular weight secondary metabolites produced in response to pathogen infection (Paxton, 1981). Phytoalexins are antimicrobial compounds that are synthesized via de novo activation of their biosynthetic pathways (González-Lamothe et al. 2009). For phytoalexins to serve in defensive response, they must be produced at the site of infection, at the correct time, and at a high enough concentration to inhibit pathogen invasion (Hammerschmidt 1999, Hammerschmidt 2011). Phytoalexins with antimicrobial activity include glyceollin from soybean (Lazarovits et al., 1980), scopoletin from tobacco and camalexin from Arabidopsis thaliana, (González-Lamothe et al. 2009). The objective of this study was to determine whether there is chemical components in the fruit peel of maturing, resistant Cucurbita moschata fruit that prevent the entry of P. capsici. This was achieved by investigating the following: 1) the presence of preformed antimicrobial compounds 53 in the fruit peel of three Cucurbita moschata cultivars at the developmental stages correlated to fruit resistance; and, 2) the production or accumulation of induced antimicrobial compounds post inoculation in the fruit peel of ‘Chieftain’ butternut squash fruit, across development stages correlated with ARR using a thin layer chromatography (TLC) bioassay. MATERIALS AND METHODS Plant material Two cultivars of butternut winter squash (Cucurbita moschata), ‘Chieftain’ and ‘Early’ and a processing pumpkin cultivar, Dickenson Field, (Table 1), previously evaluated for ARR, were used. Seeds were planted on June 20 into 72-cell flats containing a soilless peat mixture (Suremix Michigan Grower Products, Inc. Galesburg, MI) and grown in the research greenhouse on the Michigan State University (MSU) campus in East Lansing, MI, for two weeks at an average temperature of 22°C (±4°C). Plants were watered adequately as needed to maintain soil moisture. On 5 July, fifty seedlings per cultivar were transplanted into a field that was previously planted to a pumpkin at the MSU Plant Pathology Farm in Lansing, MI. The soil type was Capac loam with no known P. capsici infestation. Plants were established in raised plant beds covered with plastic mulch and irrigated twice each week via drip emitters. The plant bed length was 30.5 m with 3.7 m between rows. Twenty-five seedlings were planted into each row with two rows per cultivar. Plants were spaced 61 cm apart within the row. At flowering, the stigma of the female flowers was hand-pollinated using stamens from the male flowers and tagged. Fruits were collected 7, 10, 14, and 21 days post pollination (dpp). 54 Phytophthora capsici inoculum The virulent P. capsici isolate 12889 (mating type A1, insensitive to mefenoxam) from bell pepper (Foster and Hausbeck 2010) was obtained from the long-term stored culture collection of M. Hausbeck’s laboratory at MSU, East Lansing, MI. The culture was prepared and maintained on unclarified V8 agar (143ml V8 juice, 3g CaCO3, 16g agar, 850ml distilled water) (Dhingra and Sinclair 1985, Krasnow et al. 2017) and grown for 5 to 7 days under constant fluorescent light at room temperature. Before fruit inoculation, pathogen virulence was ensured by inoculating a cucumber fruit, recovering the pathogen, and maintaining it as previously described for use in the experiment. Non-inoculated fruits for the Thin Layer Chromatography bioassay ‘Chieftain’, ‘Early’ and ‘Dickenson’ field fruits at 10, 14, and 21 dpp were used. Three biological replicates of non-wounded fruits for each age and cultivar were surface sterilized in the laboratory by soaking the fruit in 0.4% of sodium hypochlorite solution for 5 min, rinsing with water for 2 min, and air drying on a paper towel. All fruits were peeled using a vegetable peeler; peels were placed in falcon tubes then stored at -80oC. Preparation of inoculated fruits for Thin Layer Chromatography bioassay Three non-wounded biological replicates for 7, 10, 14, and 21 dpp fruits of ‘Chieftain’ were surface disinfested as described above. Fruits were inoculated with a 7-mm agar plug from a 5- to 7-day- old P. capsici culture that was placed colonized side on the fruit at the mid-point between the peduncle and blossom end, then covered with a sterilized screw cap (16.5 mm in diameter) (Axygen Inc., Union City, CA) that was fixed to the fruit using Vaseline. Sterile V8 agar plugs 55 were used to inoculate the control fruits. Fruits were incubated in a high relative humidity chamber created by lining wet paper towels on the edges of the 99 L or 62 L plastic transparent bins (Sterilite) (Meyer and Hausbeck 2012, Krasnow et al. 2014). Throughout the incubation period, room temperature (22±2°C) and constant light were maintained. Inoculated fruits were peeled 6, 12, and 24 h post inoculation (hpi). Non-inoculated fruits were peeled following 6 h of incubation (6 h control (6hC)). All fruit peels were placed in falcon tubes then stored in the freezer at -80oC. Thin Layer Chromatography Bioassay Methodology Cladosporium cucumerinum was obtained for the long-term storage of R. Hammerschmidt’s laboratory at MSU, East Lansing, MI. Before the bioassay, cultures were grown on potato dextrose agar (PDA) for 7 to 11 d. Non-inoculated fruit peels used for analysis of the presence of antifungal compounds associated with ARR were extracted with methanol. Inoculated and non-inoculated control tissues were extracted with absolute ethanol. Ten ml of extraction solvent was used for each gram of tissue extracted. All samples were placed in a glass flask and covered with Aluminum foil. Samples were then boiled for 10 min. The tissues were re-extracted by boiling with the same solvent. All extracts were filtered through Whatman #1 filter paper. Filtrates were combined for each sample then evaporated using a rotary evaporator at 34oC. Evaporated samples were suspended in 1.0 ml of ethanol or methanol per gram fresh weight of tissue. Sonication was used to help dissolve sample extracts. All extracted samples were stored in scintillation vials in the freezer at -20oC until used in the TLC analyses. Thin Layer Chromatography (TLC) was used to separate compounds in the extracts. Silica gel G or GF TLC plates 20 x 20 cm (Analtech) were used. Samples were divided into two groups for 56 loading onto the silica plates. Group 1 included non-inoculated samples of ‘Chieftain’, ‘Early’ and ‘Dickenson Field’ 10, 14, and 21 dpp. Group 2 included inoculated ‘Chieftain’ samples 7, 10, 14, and 21 dpp at 6 hC and 6hpi. Group 3 included inoculated ‘Chieftain’ samples 7, 10, 14, and 21 dpp at 12 and 24 hpi. Fifty µl of sample was applied to TLC plates prescored to have 2 cm lanes. The plates were developed with CHCl3: methanol (9:1, v/v) to a distance of 10-13 cm. After development, the plates were allowed to dry in the fume hood before placing under vacuum in a desiccator overnight. The plates were observed using UV light (365 nm), and images were recorded using a ChromaDoc-It TLC imaging system with a Digi 105 color camera (12- megapixel), where under the UV light, compounds can be detected and characterized based on their absorbance or emission of fluorescence. Bioassay A Cladosporium cucumerinum spore suspension was prepared using 50% strength potato dextrose broth (PDB) (1.2 g PDB dehydrates in 100 ml of distilled water). PDB was added to C. cucumerinum, and mycelia were scraped to create a spore suspension that was filtered through cheesecloth mycelium and pieces of agar. The developed TLC plates were placed in a fume hood against the wall and sprayed with the prepared spore suspension until wetted thoroughly. Plates were then incubated in a humid chamber for 48 – 72 hrs. After incubation, antifungal activity was determined by the C. cucumerinum pigmented mycelia which grew across the plate except for the regions where there were compounds with antifungal activity (Klarman & Sanford, 1968). The TLC bioassay was conducted three times for the three biological replicates for each sample in the study. 57 RESULTS To determine the presence of compounds with antifungal activity in the non-inoculated fruits of ‘Chieftain’, ‘Early’, and ‘Dickenson Field’, at 10, 14, and 21 dpp, TLC bioassay was performed. The separation of the methanol extracts from the non-inoculated fruit peel samples of ‘Dickenson Field’ on cellulose plate and the cultivars Chieftain, Early, and Dickenson Field on silica G plate at 10, 14, and 21 dpp (Figures 1 and 2, respectively) showed presence of non-polar compounds through fluorescence under the UV light. Cladosporium cucumerinum grew uniformly and covered the TLC plates except for those areas with antifungal activity (Figures 1 and 2). The C. cucumerinum spores and mycelium are darkly pigmented and contrasted with the white areas on the plate representing antifungal activity. In all cultivars, TLC bioassay analyses showed the presence of compounds with antifungal activity 10, 14, and 21 dpp. Zones with antifungal activity decreased as fruit aged (Figures 1 and 2) and was consistent in all cultivars across all replicates. There was no correlation between the presence of constitutively produced compounds with antifungal activity to ARR of winter squash to P. capsici. 58 Figure 2.1.: Thin Layer Chromatography (TLC) bioassay of ‘Dickenson Field’ non-inoculated fruit peel methanol extracts at 10, 14, 21 days post pollination (dpp). Top images are methanol extract of three biological replicates, R1, R2, and R3 of ‘Dickenson Field’. The extracts were applied to a cellulose plate and developed in distilled water. Lanes 1, 2, and 3 include 25mg of 10, 14, and 21 dpp respectively. Images were captured under UV light 365nm. Bottom images are the TLC plates sprayed with Cladosporium cucumerinum and incubated for 48hrs under humidity. White areas in the red boxes refer to zones of inhibition. 59 Figure 2.2.: Thin Layer Chromatography (TLC) bioassay of ‘Early’ (lanes 1 to 3), ‘Dickenson Field’ (lanes 4 to 6), and ‘Chieftain’ (lanes 7 to 9) non-inoculated fruit peel methanol extracts at 10, 14, 21 days post pollination (dpp) respectively. Top images are sample methanol extracts applied to a silica-G-gel plate and developed in chloroform-methanol (9:1). Images were captured under UV light 365nm. Bottom images are the TLC plates sprayed with Cladosporium cucumerinum and incubated for 48hrs under humidity. White areas in the red box refer to zones of inhibition. 60 To determine if there is induced production of antifungal compounds, fruits of cultivar Chieftain were harvested at 7, 10, 14 and 21 were inoculated with P. capsici. Extracts of inoculated and control tissues were analyzed using TLC bioassay. There was a clear separation of non-polar compounds that were dissolved in ethanol for non-inoculated fruit at 6 hours (control) and inoculated with 6 hpi (Figure 3) and at 12 hpi with 24 hpi (Figure 4) under the UV light as they emitted fluorescence. The bioassay has resulted in a consistent growth of C. cucumerinum covering the entire TLC plates except for areas with antifungal compounds. The TLC bioassay revealed areas with zones of inhibition for fungal growth for all fruit ages regardless of the dpp. A significant increase in antifungal activity was not observed in the resistant fruit at 14 and 21 dpp with increased time post inoculation. A correlation between the induced production of compounds with antifungal activity and ARR was not observed. 61 Figure 2.3.: Thin Layer Chromatography (TLC) bioassay of ‘Chieftain’ inoculated fruit peel at 7, 10, 14, 21 days pos pollination (dpp) with Phytophthora capsici at 6 hours control and 6 hours post inoculation (hpi). Top images are samples ethanol extracts applied to a silica-GF-gel plate and developed in chloroform-methanol (9:1). Lanes 1 – 4 include 50mg of 7, 10, 14, and 21 dpp at 6 hours control and lanes 5 – 8 include 50mg of 7, 10, 14, and 21 dpp at 6 hours post inoculation (hpi). Images were captured under UV light 365nm. Bottom images are the TLC plates sprayed with Cladosporium cucumerinum and incubated for 48hrs under humidity. White areas in the red box refer to zones of inhibition. 62 Figure 2.4.: Thin Layer Chromatography (TLC) bioassay of ‘Chieftain’ inoculated fruit peel at 7, 10, 14, 21 days post pollination (dpp) with Phytophthora capsici at 6 hours control and 6 hours post inoculation (hpi). Top images are samples ethanol extracts applied to a silica-GF-gel plate and developed in chloroform-methanol (9:1). Lanes 1 – 4 include 50mg of 7, 10, 14, and 21 dpp at 12 hours post inoculation (hpi) and lanes 5 – 8 include 50mg of 7, 10, 14, and 21 dpp at 24 hours post inoculation (hpi). Images were captured under UV light 365 nm. Bottom images are the TLC plates sprayed with Cladosporium cucumerinum and incubated for 48hrs under humidity. White areas in the red box refer to zones of inhibition. 63 DISCUSSION ARR to P. capsici develops in some cucurbit fruits as they mature. Understanding the mechanisms of this form of resistance may be helpful in integrated management strategy to limit fruit rot disease. In the current study, the possible role of antifungal compounds in ARR to P. capsici was examined. ARR phenomenon has also been observed in several plants against fungi, bacteria, viruses, and oomycetes (González-Lamothe et al. 2009). The mechanism of ARR seems to be variable among different plant systems (Panter and Jones 2002). Hypocotyl tissue of cucumber and soybean seedlings become resistant to Cladosporium cucumerinum (Panter and Jones 2002, Hammerschmidt 2011) and Phytophthora sojae (Lazarovits et al. 1980), respectively, as they age. Arabidopsis plants develop ARR to Pseudomonas syringae pv. tomato (Kus et al. 2002) and cauliflower mosaic virus (Leisner et al. 1993). The mechanism of ARR associated with fruit maturation has been studied in cucumber and pepper (Ando et al. 2015, Biles et al. 1993). One of the potential mechanisms of plant defense against pathogens is the production of defensive secondary metabolites with antimicrobial activity (González-Lamothe et al. 2009). These defensive phytochemicals can be produced before pathogen infection and stored in plant tissues (i.e., phytoanticipins), or synthesized de novo (i.e., phytoalexins) in response to a pathogen attack (VanEtten et al. 1994, Paxton 1981). Our results revealed a decrease in constitutively produced antimicrobial activity in the non-inoculated fruit peel as the fruit aged (Figures 2.1, 2.2). A significant change in the antimicrobial activity following P. capsici infection was not observed (Figures 2.3, 2.4). Our findings did not support the hypothesis that a chemical barrier to P. capsici in resistant maturing fruit is correlated with ARR in C. moschata cultivars. 64 The role of phytoalexins as defensive antimicrobial compounds has been studied in several plants such as camalexin, from Arabidopsis and Camelina. Infection of Arabidopsis with fungi, bacteria, viruses, and oomycetes induces the production of camalexin (González-Lamothe et al. 2009). High concentrations of camalexin were detected at the infection sites of Arabidopsis in association with infection by Alternaria alternate (Schuhegger et al. 2007). In Cucurbita fruit, trans-p- coumaryl aldehyde, a lignin-like material, was identified in C. maxima squash fruit tissue elicited with pectinase. The antifungal activity of this compound was confirmed using TLC-bioassay; p- coumaryl aldehyde was also detected in other elicited tissue of butternut, cucumber and pumpkin fruits (Stange et al. 1999). This compound was also induced by C. cucumerinum in scab-resistant cucumber seedlings (Varbanova et al. 2011). Also, C-glycosyl flavonoids have shown to function as phytoalexins in cucumber leaves elicited with Milsana and inoculated with powdery mildew fungus. A role for phytoanticipins as plant defensive secondary metabolites with antimicrobial activity against pathogens have been demonstrated in several plant-pathogen systems (Panter and Jones 2002, González-Lamothe et al. 2009). Production of avenacin A-1 in oats roots has been shown to be a factor in resistance of that host to Gaeumannomyces graminis var. tritici (Osbourn et al. 1994). In a cucumber variety with ARR to P. capsici, the methanol extract of resistant maturing fruit peel was detected to inhibit P. capsici growth in vitro (Ando et al. 2015). When this methanol extract was metabolically analyzed, enrichment of terpenoid glycosides was detected (Mansfeld et al. 2017), which suggested a potential correlation of the constitutively produced secondary metabolite with the observed ARR in cucumber fruit. Although cucumber is in the same family Cucurbitaceae as squash, it seems that different mechanisms are controlling ARR in the different genera. 65 In the cucurbit, rockmelon (Cucumis melo L), resistance to Fusarium rot caused by F. oxysporum f. sp. melonis was associated with immature fruit; susceptibility increased with fruit age (Kumar and McConchie 2010). Kumar and McConchie (2010) detected two preformed antifungal compounds in rockmelon fruit peel, and their antifungal activity decreased with fruit age. In our study, a decrease in antifungal activity was detected and diminished with age. The reduction in antifungal activity in fruit peel correlated with ARR detected in rockmelon but was not detected in C. moschata cultivars. This study did not provide evidence for constitutive or induced antifungal compounds as part of ARR P. capsici in winter squash. A study of fruit exocarp structural changes in ‘Chieftain’ butternut squash across development has shown that cuticle and epidermal walls were significantly thicker in resistant maturing fruit at 14 and 21 dpp than susceptible 7 dpp fruit, which suggests the presence of a structural barrier to P. capsici infection of the resistant fruit (Alzohairy et al. 2017). Cuticle thickness also increased with fruit age in pepper with ARR to P. capsici (Biles et al. 1993). While the mechanism of ARR can be structural or chemical, our study did not show a correlation between the production of either constitutive or induced antifungal activity and ARR to P. capsici. These results may rule out a biochemical defense associated with ARR of C. moschata fruits and suggests that other structural or genetic factors control ARR. 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Plant Physiology, 157:1056-1066. 71 CHAPTER III: TRANSCRIPTOMIC PROFILING OF WINTER SQUASH IMPLICATES MONOLIGNOLS BIOSYNTHESIS AND LIGNIN POLYMERIZATION IN AGE-RELATED RESISTANCE TO THE OOMYCETE PHYTOPHTHORA CAPSICI ABSTRACT The oomycete plant pathogen, Phytophthora capsici, causes root, crown, and fruit rot of winter squash (Cucurbita spp.) and limits production. Some Cucurbita moschata cultivars develop age- related resistance (ARR) whereby fruit develop resistance to P. capsici 21 days post pollination (dpp) due to thickening exocarp, cuticle, and epidermal walls; wounding negates ARR. To determine the molecular mechanisms of ARR two C. moschata cultivars that exhibit ARR at different dpp were chosen and the transcriptome of fruit peel at susceptible and resistant time points was sequenced. Upregulated genes in the fruit peel resistant to P. capsici in both cultivars tended to play roles in cell wall and lignin biosynthesis and possess peroxidase and cinnamyl alcohol dehydrogenase activities. In addition, cutin, suberin, and phenylpropanoid biosynthetic pathway genes tended to be consistently upregulated when comparing resistant to susceptible time points. Our results provide candidate genes essential for C. moschata resistance to P. capsici, enabling the development of C. moschata varieties with resistance to P. capsici for improvement of squash and pumpkin fruit rot management schemes. 72 INTRODUCTION Phytophthora capsici is a soilborne oomycete with a host range exceeding 50 plant species (Tian and Babadoost 2004). This polycyclic pathogen is responsible for significant plant losses when environmental conditions are favorable (Erwin and Ribeiro 1996, Hausbeck and Lamour 2004, Granke et al. 2009). Economically important crops within the Cucurbitaceae, Solanaceae, and Fabaceae are highly susceptible to P. capsici infection (Davidson et al. 2002, Hausbeck and Lamour, 2004, Gevens and Hausbeck 2005); cucurbits are considered among the most susceptible (Tian and Babadoost 2004). Symptoms of P. capsici infection on cucurbits include root and/or crown rot, foliar blight, and fruit rot (Babadoost 2004). Fruit rot threatens cucurbit crops annually, including squash and pumpkin, in Michigan (Lamour and Hausbeck 2000, Gevens et al. 2007, Krasnow and Hausbeck 2016) and other states (Babadoost 2004, Castro-Rocha et al. 2017). Michigan is an important producer of summer and hard squash (USDA 2018). The fruits may become rotted while in the field (Meyer and Hausbeck 2013, Granke et al. 2012) or postharvest (Hausbeck and Lamour 2004) leading to loss in crop production that may exceed 50% (Babadoost 2000, Meyer and Hausbeck 2013). Protecting squash fruits from P. capsici infection is challenging due to a relatively lengthy maturation time and during which the fruits are in direct contact with the soil. While host resistance is critical for long-term management (Quesada and Hausbeck 2010, Granke et al. 2012), complete host resistance in commercial cultivars of squash or pumpkin is not available (Café-Filho et al. 1995). However, age-related resistance (ARR) to P. capsici is expressed in the fruits of specific C. moschata cultivars (Meyer and Hausbeck 2013, Krasnow and Hausbeck 2016) and other cucurbit (Gevens et al. 2006, Ando et al. 2009) and Solanaceae (Biles et al. 1993) fruit. ARR is associated with resistance to pathogens that occurs at specific developmental stages (Stermer and Hammerschmidt 1984, Whalen, 2005). Fruits of a number of 73 cucurbits (Gevens et al. 2006, Ando et al. 2009, Meyer and Hausbeck 2013, Krasnow and Hausbeck 2016) and pepper (Biles et al. 1993) exhibit ARR to P. capsici as they mature. However, wounding negates ARR to P. capsici (Ando et al. 2015, Biles et al. 1993, Krasnow et al. 2014), suggesting the fruit peel may provide resistance to fruit rot. The mechanism of ARR has been investigated in different host-pathogen systems (Panter and Jones 2002, Develey-Rivière and Galiana 2007). ARR can be conferred by preformed or induced defenses (Panter and Jones 2002, González-Lamothe et al. 2009) where preformed defense is the consequence of structural/physical and/or chemical barriers (Vergne et al. 2010). The plant cell wall serves as a physical barrier that forms an obstacle for all pathogens’ entry but can be overcome by pathogen-generated cell wall degrading enzymes (CWDE) (Bacete et al. 2018, Bellincampi et al. 2014). In addition to cell wall, the plant surface is covered by the cuticle made up of cutin polymer that provides another defensive layer against pathogens (Chassot and Métraux 2005). Thickening of bean hypocotyls is correlated with resistance to Rhizoctonia solani (Stockwell and Hanchey 1983). Similarly, thickening of the cuticle has been suggested as the mechanism of ARR in pepper fruit (Biles et al. 1993) and C. moschata cultivars (Alzohairy et al. 2017) to P. capsici. Thickening of the epidermal walls was also observed in cucumber fruit with ARR to P. capsici (Ando et al. 2015). Another form of constitutive defense involving strengthened physical barrier is the formation of lignified xylem vessels in bean that leads to resistance against Colletotrichum lindemuthianum (Griffey and Leach 1965). In addition, lignin deposition at the cell wall makes it resistant to CWDE as has been observed in resistance of Camelina sativa to Sclerotinia sclerotiorum (Eynck et al. 2012). However, it remains to be determined if increased ligin deposition is important for ARR in cucurbits. 74 Transcriptomic studies of cucumber fruit with ARR showed an increase in the level of terpenoid glycosides in resistant maturing cucumber fruit compared to the susceptible younger fruit suggesting a role of constitutive chemical defense (Mansfeld et al. 2017). When the molecular mechanisms controlling ARR in apple leaves to Venturia inaequalis was studied, the constitutive upregulation of genes encoding for metallothionein3-like protein, lipoxygenase, lipid transfer protein, and peroxidase 3, and downregulation of gene encodes for ‘enhanced disease susceptibility 1 protein’ were highly correlated with the observed ARR of aging apple leaves (Gusberti et al. 2013). Previous studies of fruit ARR in squash and pumpkin against P. capsici infection attribute ARR to morphological (Ando et al. 2009) or physiological changes (Meyer and Hausbeck 2013). The different developmental time points of ARR onset have been observed in cultivars of C. moschata (Alzohairy et al. 2017) and other cucurbits (Ando et al. 2009, Gevens et al. 2006). The difference in ARR onset has been suggested to be related to the difference in rates of fruit development (Gevens et al. 2006). However, the genetic mechanism of ARR in winter squash and how the difference in ARR is regulated across cultivars is not known. This study aims to use transcriptomic studies and differential gene expression analysis between resistant and susceptible fruit stages to assess the molecular mechanisms of ARR in two C. moschata cultivars with different ARR onset time points. MATERIALS AND METHODS Plant material Two Cucurbita moschata commercial cultivars, Chieftain (butternut winter squash, seeds were obtained from Rupp Seeds Inc., Wauseon, OH) and Dickenson Field (processing pumpkin, seeds 75 were obtained from Rispens seeds Inc.), were previously evaluated for age-related resistance (ARR) (Meyer and Hausbeck 2013, Alzohairy et al. 2017). Fruits of these two cultivars were produced in the field according to Meyer and Hausbeck (2013). At anthesis, flowers were hand pollinated then fruits were harvested at 7, 10, 14, and 21 days post pollination (dpp). RNA extraction Fruits of ‘Chieftain’ and ‘Dickenson Field’ at 7, 10, 14, and 21 dpp were surface disinfested with 70% ethanol then air-dried on paper towel. Sterilized fruits were peeled using the vegetable peeler and immediately frozen in liquid nitrogen. Fruit peels were stored at -80°C until RNA isolation. RNA was extracted from three biological replicate samples for each cultivar and each date using E.Z.N.A. Total RNA kit (OMEGA BIO-TEK) and treated with Turbo DNase (2U/µl, Invitrogen) to remove DNA contamination. RNA concentration was determined using Nanodrop 1000 spectrophotometer (Thermo Scientific). RNA integrity (RIN) was checked using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All samples had RIN scores ≥8. RNA sequencing and RNA-seq read processing RNA library construction and sequencing were done at the Research Technology Support Facility (RTSF) at Michigan State University. RNA libraries were prepared using the Illumina TruSeq Stranded mRNA kit following the Illumina protocols. RNA sequencing was performed using the Illumina HiSeq 4000 platform. Illumina run was in the 2 x 150bp paired-end format. A total of 24 libraries were divided into two pools of 12, and each pool was sequenced on two lanes. Each lane produced an approximate of 30 million reads/sample. Due to a technical error in the sequencing run, two libraries for ‘Chieftain’ at 14 dpp were resequenced on the same platform, which produced ~180 million read/sample. 76 An initial quality check of the RNA reads was performed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). RNA reads from the two lanes for the same sample were combined in a single fastq file for each R1 and R2. Trimmomatic (Bogler et al. 2014) v0.32 was used to filter the RNA reads for adaptors, and low-quality reads. Sliding window method was used to scan the reads with 4-base wide and cut when the base quality is below a threshold of 2. The minimum read length cutoff was 20 bp. Data quality was explored after filtering with FastQC. STAR v2.5.1b (Spliced Transcripts Alignment to a Reference, Dobin et al. 2013) was used to map the RNAseq reads to the Cucurbita moschata cv. Rifu reference genome (Sun H et al. 2017) from the Cucurbit Genomics Database (http://cucurbitgenomics.org/organism/9). STAR was used with the default settings using the twopassMode Basic option with intron size 21-6000 nt. In all samples, >88% of the RNAseq reads were mapped to the reference genome. The number of sequenced reads, filtered reads, and mapping information are presented in Appendix Table 3. 1. Differential expression and clustering analysis The HTseq-count function in HTseq (High-Throughput sequencing) (Anders et al. 2015) v0.6.1 was used in the default mode and stranded==reverse for generating read counts. HTseq-count output was fed into DESeq2 (Love and Anders 2014) for differential expression analysis using the standard steps represented in DESeq function (Love et al. 2017). A gene was considered differentially expressed in contrast if the adjusted p-value < 0.05 and the log fold change > 1. The p-value was adjusted with Benjamini-Hochberg. DEGs from all comparisons among cultivars and were categorized into three main sets including set1: genes that are upregulated or downregulated among all tested comparisons across cultivars; set2: genes that 77 are commonly upregulated or downregulated in all comparisons per cultivar and set3: genes that are expressed at least in one comparison in both cultivars (Supplementary Table 1). The clustering of expression data was done using k-means clustering with the Complex Heatmap package in R (Gu. et al., 2016) with k = 16. Identification of putative Arabidopsis orthologous genes and inference of squash cell well pathway genes Putative orthologous Arabidopsis thaliana genes of C. moschata DEGs and non-DEGs were identified using BLASTX (Altschul et al. 1990) by comparing the nucleotide sequence of the C. moschata DEGs and non-DEGs to the peptide sequence of A. thaliana using e-value of 10e-10. Arabidopsis pathways related to cell wall structure/composition were downloaded from (https://www.plantcyc.org/). The pathways include cuticular wax biosynthesis, cutin biosynthesis, long-chain fatty acid activation, suberin monomers biosynthesis, esterified suberin biosynthesis, cellulose biosynthesis, homogalacturonan biosynthesis, xylogalacturonan biosynthesis, phenylpropanoid biosynthesis, and xylan biosynthesis. Functional annotation and pathway enrichment analysis All DEGs and non-DEGs were functionally annotated through extracting the Gene Ontology (GO) annotations, Interpro, and description from Cucurbit Genomics database (http://cucurbitgenomics.org/organism/9). GO enrichment analysis were done for the different DEGs lists for both cultivars independently. Pathway enrichment analysis were performed using the C. moschata genes orthologous to the Arabidopsis genes in ten targeted biosynthesis pathways, which are cuticular wax, cutin, long chain fatty acid activation, suberin monomers, esterified 78 suberin, cellulose, homogalacturonan, xylan, xylogalacturonan, phenylpropanoid. Both GO and pathway enrichment analysis were based on Fisher’s exact tests. p-values were corrected for multiple testing (Benjamini and Hochberg 1995) and reported as q-values. DEGs were considered significantly overrepresented for a GO-term or pathway when they were positively enriched, and their q-value was <=0.05. Fisher’s exact tests were performed using manual python scripts which employ the python package fisher 0.1.5 https://pypi.org/project/fisher/ . RESULTS Sequencing and gene expression profile among cultivars To study ARR in winter squash to P. capsici, the fruit peel transcriptomes of two cultivars, Chieftain and Dickenson, at different developmental time points prior to and after the development of ARR were sequenced (Figure 3.1A). C. moschata genes were classified into 16 clusters using the transcriptome of both cultivars at different developmental time points to assess the extent to which gene expression patterns correlated with ARR onset (Figure 3.1B). These clusters were grouped into 4 groups based on the similarity in gene expression profile during different time points and between cultivars. We anticipated that ARR-associated genes would have an expression profile that consists of two major patterns. First, groups of genes with a consistent change of expression either by upregulation or downregulation in both cultivars in at least one resistant time point (Figure 3.1B, groups 1 and 2). Second, groups of genes whose expression were upregulated or downregulated in at least one resistant stage in only one cultivar (Figure 3.1B, groups 3 and 4). Groups 3 and 4 represent the opposite expression pattern in both cultivars. For example, in one cultivar, genes may be upregulated in at least one susceptible stage then become downregulated in the resistant stages. This pattern was reversed in the second cultivar in the same cluster. Also, 79 groups 3 and 4 include genes with irregular expression pattern (e.g. genes can be going up-down- up-down through the four stages of each cultivar). Genes in groups 3 and 4 are less likely to be to be candidates for ARR. Dividing the genes into different clusters facilitated identifying a group of genes that are likely candidates of ARR (Figure 3.1B, groups 1 and 2). Therefore, we performed differential expression analysis to detect potential ARR-associated genes. A B I Figure 3.1.: A) Expression conditions of cultivars Chieftain and Dickenson fruit peel at ages 7, 10, 14, and 21 days post pollination. B) Heatmap of K-means clustering of per-gene normalized expression between both ‘Chieftain’ and ‘Dickenson’ at 7, 10, 14, 21 dpp. Numbers 1, 2, 3, and 4 are different groups of clusters. Genes were clustered at K=16. 80 Differential gene expression of ‘Chieftain’ and ‘Dickenson Field’ In ‘Chieftain’, ARR develops at 14 dpp and continues through 21 dpp, while ARR in ‘Dickenson’ develops at 21 dpp. To identify candidate genes relevant to resistance development at these ages in ‘Chieftain’, we contrasted the gene expression of the susceptible fruit peels at 7 and 10 dpp to the resistant ones at 14 and 21 dpp. Similarly, gene expression of the susceptible fruit peels was contrasted at 7, 10, and 14 dpp with the resistant 21 dpp in ‘Dickenson’. Candidate genes responsible for resistance against P. capsici included genes significantly upregulated or downregulated at 14 and 21 dpp in ‘Chieftain’ and at 21 dpp in ‘Dickenson’ when contrasted to their susceptible fruit peel ages (Figure 3.2A). The overlap between the different contrasts per each cultivar for both upregulated (Figure 3.2A, B, C) and downregulated genes (Figure 3.2A, D, E) was used to identify DEGs that were consistently upregulated or downregulated in the resistant stages compared to their susceptible stages. When comparing the resistant stages in ‘Chieftain’ to the susceptible stages, DEGs tended to be similarly up- or down-regulated. Thus, there are groups of genes with expression patterns that are correlated with the resistant phenotype (Figure 3.2A). In addition, fewer DEGs were detected when comparing 14 and 21 dpp to 10 dpp than to 7 dpp (Figure 3.2B, D) and that difference in DEGs helped to narrow down the list of DEGs that are likely candidate genes for ARR. Similarly, in ‘Dickenson’, there was an apparent decrease in the number of the determined DEGs as the fruit aged toward the resistance showing also a gradual transition toward resistance that developed at 21 dpp (Figure 3.2C, E). From the differential gene expression analysis, we narrowed down the DEGs to sets 1, 2, and 3 that are potentially included in the ARR-associated genes. 81 A I Figure 3.2.: Differential gene expression analysis of ‘Chieftain’ and ‘Dickenson’ fruits at 7, 10, 14 and 21 days post pollination (dpp). The onset of age-related resistance (ARR) is at 14 and 21 dpp in ‘Chieftain’ and ‘Dickenson’, respectively. A) Heatmap of differentially expressed genes (DEGs) with |log2(FC)|>1, (FC: fold change) and adjusted p-values <0.05. Each column is a contrast between resistant vs susceptible peel of fruit at 7, 10, 14 and 21 dpp. A) and B) Venn diagrams showing upregulated and downregulated genes in all sets of comparisons in ‘Chieftain’ respectively; C) and D) Venn diagrams showing upregulated and downregulated genes in all sets of comparisons in ‘Dickenson’, respectively. The letters C and D indicates ‘Chieftain’ and ‘Dickenson’, respectively. 82 Figure 3.2.: (cont’d) B C D E I 83 Function of upregulated genes in both cultivars The function of the upregulated genes in the resistant fruit peel stages when contrasted to their susceptible stages was investigated to determine which genes are candidates for controlling ARR. In ‘Chieftain’, we included the resistant stages of 14 and 21 dpp to determine if there are stage- specific genes that could be related to ARR versus those that may be constant during both 14 and 21 dpp. Therefore, the Gene Ontology (GO) categories enriched in upregulated genes resulted from individual contrasts were identified. First, the function of the upregulated genes that were detected from the contrast between 14 dpp to 7 and 10 dpp were investigated in order to detect the functions that were consistently present in the resistant stage 14 dpp and related to ARR. Among 3226 and 1120 upregulated genes in ‘Chieftain’ resulting from the contrast of the early resistant time point (14 dpp) vs. susceptible time points (7 and 10 dpp), they were enriched in 33 and 44 GO-terms, respectively (Appendix Table 3.2). The most significantly overrepresented GO terms during early resistant time point include those relevant to cell wall structures (e.g. lignin biosynthesis process) and phenylpropanoids biosynthesis (e.g. cinnamyl alcohol dehydrogenase activity, sinapyl alcohol dehydrogenase activity), oxidoreductases (e.g. peroxidase activity) and defense (e.g. defense response to bacterium) (Figure 3.3, Appendix Table 3. 2). This result indicates that a group of genes functioning in cell wall structures and phenylpropanoid biosynthesis are likely related to ARR at 14 dpp fruit in ‘Chieftain’. 84 Figure 3 s m r e t O G neg.log.qval/log.qval 2 1 0 −1 −2 oxidation reduction process peroxidase reaction coumarin biosynthetic process lignin biosynthetic process stilbene biosynthetic process oxidoreductase activity heme binding cinnamyl alcohol dehydrogenase activity sinapyl alcohol dehydrogenase activity peroxidase activity transcription factor complex defense response to bacterium hydrogen peroxide catabolic process chlorophyll binding photosystem II photosynthesis photosystem I photosynthetic electron transport in photosystem I chlorophyll biosynthetic process stomatal complex morphogenesis regulation of meristem growth root morphogenesis response to chitin defense response to fungus detection of biotic stimulus response to biotic stimulus 7 C _ s v _ p u 4 1 C 0 1 C _ s v _ p u 4 1 C 7 C _ s v _ p u 1 2 C 7 D _ s v _ p u 1 2 D 0 1 C _ s v _ p u 1 2 C 0 1 D _ s v _ p u 1 2 D 4 1 D _ s v _ p u 1 2 D 3 t e S n w o d _ 1 e S t p u _ 1 t e S i i i n a t f e h C _ p u _ 2 e S n o s n e k c D _ p u _ 2 e S t t 7 C _ s v _ n w o d 4 1 C 7 C _ s v _ n w o d 1 2 C 0 1 C _ s v _ n w o d 4 1 C 7 D _ s v _ n w o d 1 2 D 0 1 C _ s v _ n w o d 1 2 C 0 1 D _ s v _ n w o d 1 2 D 4 1 D _ s v _ n w o d 1 2 D i i i n a t f e h C _ n w o d _ 2 e S n o s n e k c D _ n w o d _ 2 e S t t upregulated downregulated Comparisons Figure 3.3.: Heatmap showing selected GO terms that are significantly overrepresented in either upregulated genes or downregulated genes in contrasts between resistant vs susceptible peel of fruit at 7, 10, 14 and 21 days post pollination (dpp) in both ‘Chieftain’ and ‘Dickenson’. The onset of age-related resistance (ARR) is at 14 and 21 dpp in ‘Chieftain’ and ‘Dickenson’ respectively. The letters C and D indicates for ‘Chieftain’ and ‘Dickenson’ respectively. The value range of the heatmap is shown as the result of the Fisher’s Exact test. If the GO term was overrepresented, the negative log of the adjusted p-value (or q-value) was taken, while if it was underrepresented, the log of the adjusted p-value was taken. Therefore, a positive value > or = 1.3 indicates significant overrepresentation while a negative value < or = -1.3 indicates significant underrepresentation. The black rectangles point to functions related to cell wall structures and phenylpropanoid biosynthesis processes that are overrepresented only in the upregulated genes in both cultivars. Next, we studied the function of the upregulated genes detected from contrast between late resistant stage 21 dpp and susceptible stages (7 and 10 dpp) in ‘Chieftain’. By defining the function of the upregulated genes in late resistant stage (21 dpp), the functions that are related to ARR can be detected by filtering the shared functions with early resistant stage (14 dpp). Also, comparing the functions that are detected in both early (14 dpp) and late resistant (21 dpp) stages will define 85 differences in functions that could be related to each resistant time point. Among 2047 and 1482 ‘Chieftain’ upregulated genes when comparing expression levels during the late stage of resistance (21 dpp) to those during susceptible time points 7 and 10 dpp, they were enriched in 38 and 41 GO terms respectively (Appendix Table 3.2). Similar GO terms were detected between early (14 dpp) and late (21 dpp) resistant stages include cell wall structures (e.g. lignin biosynthesis process) and phenylpropanoids biosynthesis (e.g. cinnamyl alcohol dehydrogenase activity, sinapyl alcohol dehydrogenase activity), oxidoreductases (e.g. peroxidase activity), and defense (e.g. defense response to bacterium) (Figure 3, Appendix Table 3. 2). Few GO terms were different between 14 and 21 dpp when compared to 7 and 10 dpp include GO terms relevant to fruit ripening (e.g. xylem development) and sugar hydrolysis (e.g. beta-galactosidase, glycosaminoglycan catabolic process) (Appendix Table 3. 2). The different GO terms between 14 and 21 dpp did not indicate specific enrichment in functions relevant to resistance (Appendix Table 3. 2), suggesting that the resistance is most likely associated with genes that are consistently upregulated in both 14 and 21 dpp in contrast to the susceptible stage 7 and 10 dpp. In ‘Dickenson’ the functions of upregulated genes detected in the contrasts of 21 vs.7 dpp, 21 vs.10 dpp, and 21 vs.14 dpp were enriched in 45, 43, 48 GO terms respectively (Appendix Table 3. 2). Overrepresented GO terms most relevant to the resistance are similar to those detected in ‘Chieftain’ contrasts included cell wall structures and phenylpropanoids biosynthesis (Figure 3.3). Cultivars distinct GO terms were not related to resistance (Appendix Table 3. 2). Taken together, group of GO terms were identified that were consistently enriched in the upregulated genes detected in the resistant stages in both cultivars when contrasted to their susceptible stages, suggesting that the genes involved in these functions are likely to be ARR-associated genes. 86 Function of downregulated genes in both cultivars To identify the genes that are downregulated during fruit development and the transition to a resistant state, the function of the detected downregulated genes in the resistant stages in both cultivars using GO was assessed. In ‘Chieftain, among the 4628 and 1688 downregulated genes in 14 vs. 7 dpp and vs. 10 dpp, 127 and 149 GO terms were enriched, respectively. Among the 2896 and 1792 downregulated genes at 21 dpp when compared to 7 and 10 dpp, 152 and 161 GO terms, respectively, resulted (Appendix Table 3.3). These GO terms are highly overrepresented in photosynthesis (e.g. chlorophyll binding, photosystem II, chloroplast thylakoid membrane), cell growth (e.g. regulation of meristem growth, tissue development, regulation of cell size), cell differentiation (e.g. stomatal complex morphogenesis, root morphogenesis) and defense (e.g. defense response to fungi or bacteria, negative regulation of defense) (Figure 3.3, Appendix Table 3.3). This result indicates that in ‘Chieftain’, ARR associated genes are genes that are upregulated in the resistant stages in comparison to the susceptible stages. In ‘Dickenson’, the 4644, 3577, and 1284 downregulated genes in the contrasts of 21 dpp vs. 7, 10, and 14 dpp were enriched for 165, 167, and 170 GO terms respectively (Appendix Table 3. 3). Shared categories of GO terms among the three different contrasts of ‘Dickenson’ are overrepresented with high significance in photosynthesis (e.g. photosystem I and II, plastid organization, chloroplast thylakoid membrane), and metabolic processes (e.g. sucrose and starch metabolic process), in addition to other GO terms categories such as defense (e.g. systemic acquired resistance, detection to biotic stimulus, defense response to fungus) (Figure 3.3, Appendix Table 3. 3). This result indicates that in ‘Dickenson’, ARR associated genes are in the upregulated list of genes in the resistant fruit 21 dpp. Our findings from the functional annotation 87 of both upregulated and downregulated genes detected in the comparisons of resistant vs. susceptible fruit ages in both cultivars indicate that the mechanism of ARR is most likely controlled similarly in both cultivars. Comparison of ARR mechanism among cultivars The studied cultivars of C. moschata have a different onset of ARR to P. capsici, with ‘Chieftain’ developing resistance at 14 dpp and ‘Dickenson’ developing resistance at 21 dpp. To determine whether the mechanism of ARR to P. capsici is similar or different across cultivars, DEGs between the two cultivars and among the different developmental stages were categorized into three sets of genes (Figure 3.2A, Supplementary Table 1). The Set1 genes included those that are consistently upregulated or downregulated in both cultivars in the resistant fruit peel ages in comparison to the susceptible fruit peel ages (Figure 3.2A). The Set1 genes are likely to be genes that are associated with resistance in case the mechanism of ARR is similar acoss cultivars. The Set2 genes are commonly upregulated or downregulated in all comparisons within each cultivar. The Set2 genes are likely to be cultivar specific genes, and therefore, we can determine if the mechanism is different between cultivars. Finally, the Set3 genes are those that are either up or down-regulated in at least one contrast between resistant and susceptible stages in both cultivars (Figure 3.2A). The Set3 genes can be related to the resistance mechanism that could be specific to any resistant stage in both cultivars. Upregulated genes in Set1 were enriched in 38 GO terms (Appendix Table 3. 2) and the overrepresented GO terms included cell wall structures, phenylpropanoid biosynthesis (e.g. lignin biosynthesis process, sinapyl alcohol dehydrogenase activity, cinnamyl alcohol dehydrogenase activity, stilbene biosynthetic process, and coumarin biosynthetic process), and oxidoreductases 88 (e.g. peroxidase activity). In Set2, upregulated genes in ‘Chieftain’, were enriched in 40 GO terms. The top ten significantly overrepresented GO terms included the oxidoreductase activity, stilbene biosynthetic process, sinapyl alcohol dehydrogenase activity, and cinnamyl alcohol dehydrogenase activity (Appendix Table 3. 2). However, other overrepresented GO terms for lignin biosynthetic process, peroxidase activity, and coumarin biosynthetic process were still detected (Appendix Table 3. 2). Upregulated genes of Set2 in ‘Dickenson’ were enriched in 49 GO terms with the highly scored including sinapyl alcohol dehydrogenase activity, cinnamyl alcohol dehydrogenase activity, lignin biosynthetic process, peroxidase activity, and coumarin biosynthetic process (Appendix Table 3. 2). We also questioned if the ARR phenotype is correlated with genes that are expressed in any comparison in both cultivars, Set3. The function of DEGs in Set3 were enriched in 29 overrepresented GO terms but didn’t seem to be relevant to ARR (Appendix Table 3. 2 and 3. 3). We also investigated the function of downregulated genes detected in all different sets. Downregulated genes in Set1, Set2 in Chieftain, and Set2 in Dickenson were enriched in 114, 154, and 174 GO terms respectively (Appendix Table 3. 3). The highly-scored GO terms for all sets were involved in photosynthesis (e.g., chlorophyll binding, protein chromophore linkage, photosynthesis, chloroplast thylakoid membrane and photosystem I and II) (Figure 3, Appendix Table 3. 3). According to these findings we sought to question which cell wall structures are specifically regulated during development and can be a candidate for the winter squash ARR mechanism against P. capsici. 89 Pathway enrichment of cell wall structure-related genes In the GO enrichment analysis, we detected that cell wall structures and phenylpropanoid biosynthesis processes are enriched in the upregulated genes in the resistant stages in both cultivars. Based on this finding and also the findings from Alzohairy et al. 2017 that cuticle and epidermal walls thickness increase in the resistant fruit ages so we hypothesized that cell wall structures biosynthesis is related to ARR. To define which cell wall structure is related to ARR we studied the enrichment of different cell wall structures biosynthesis pathways in the lists of upregulated and downregulated genes detected in the peel of resistant fruit ages in both cultivars. The winter squash orthologous upregulated and downregulated genes in the resistant stages were examined for enrichment in pathways for cell wall structures biosynthesis. First, the nucleotide sequences of upregulated and downregulated genes in the resistant stages were blasted against Arabidopsis peptide database and orthologous squash genes were detected. Then, orthologous squash genes to Arabidopsis genes that are involved in ten different cell wall structures biosynthesis pathways were used in the enrichment analysis. Out of ten tested pathways, five were enriched in upregulated and downregulated genes in the resistant fruit stages in both cultivars, which are cutin, phenylpropanoid, suberin monomers, homogalacturonan, and cellulose biosynthesis (Figure 3.4). We detected that cellulose and homogalacturonan biosynthetic pathways were overrepresented in the lists of downregulated genes in resistant stages of both cultivars. While cutin biosynthetic pathway was overrepresented in both lists of up and downregulated genes in both cultivars but more in the downregulated lists of genes in the case of ‘Chieftain’ (Figure 3.4). Suberin monomers and phenylpropanoid biosynthesis pathways were overrepresented in the upregulated lists of genes for both cultivars more than in the downregulated lists of genes (Figure 3.4). 90 upregulated downregulated Figure 3.4.: Heatmap showing the pathway enrichment analysis of hard squash of C. moschata orthologous genes in Arabidopsis cell wall structures biosynthetic pathways. Columns show the contrast between resistant vs susceptible peel of fruit at 7, 10, 14 and 21 days post pollination in both ‘Chieftain’ and ‘Dickenson’. The onset of age-related resistance (ARR) is at 14 and 21 in ‘Chieftain’ and ‘Dickenson’ respectively. The letters C and D indicates for ‘Chieftain’ and ‘Dickenson’ respectively. The value in the range of the heatmap is shown as the results of the Fisher’s Exact test. If the pathway was overrepresented, the negative log of the adjusted p-value (or q-value) was taken, while if it was underrepresented, the log of the adjusted p-value was taken. Therefore a positive value > or = 1.3 indicates significant overrepresentation while a negative value < or = -1.3 indicates significant underrepresentation. For a better understanding of the enrichment patterns in upregulated versus downregulated genes, we plotted the expression profile of all orthologous upregulated and downregulated genes in the resistant fruit stages that were detected from the different contrasts in both cultivars and involved in phenylpropanoid, suberin monomers, cutin, cellulose, and homogalacturonan biosynthetic pathways (Figure 3.5A, B, C, D, E). The general expression profile of squash orthologous genes 91 showed more upregulated genes associated with the phenylpropanoid, suberin monomers, and cutin biosynthetic processes (Figure 3.5A, B, C) while downregulated genes were more associated with the cellulose and homogalacturonan biosynthetic pathways (Figure 3.5D, E). These results indicate that the phenylpropanoid, suberin monomers, and cutin biosynthetic processes are most likely associated with the observed ARR since they are enriched in the upregulated genes in the resistant stages in both cultivars. 92 A B C D E Figure 3.5.: General expression profile of squash orthologous genes that are differentially expressed (p<0.05, Log2(FC) >1, FC: Fold Change) in the different contrasts in both cultivars and involved in A) phenylpropanoid, B) suberin monomers, C) cutin, D) cellulose, and E) homogalacturonan biosynthetic pathways. 93 To determine which genes are highly associated with the ARR of winter squash fruit peel, a few upregulated genes from the three pathways (cutin, suberin monomers and phenylpropanoid) were selected and their expression in both cultivars (Figure 3.6A, B, C) was plotted. Genes encoding for Acyl COA thioesterases and long-chain fatty acid CoA ligase (LACs) involved in cutin biosynthesis pathway were upregulated in both cultivars (Figure 3.6A). Three genes encoding for phenylalanine ammonia-lyase (PAL), caffeoyl-CoA O-methyltransferase (cCoAOMT), and p- coumarate 4-hydroxylase (C4H) involved in suberin monomers biosynthesis pathway (cytochrome P450 protein) were upregulated in the resistant stages in each cultivar (Figure 3.6B). Two genes encoding for cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR) (Figure 3.6C) associated with phenylpropanoid biosynthesis pathway were consistently upregulated in all resistant fruit peel ages in both cultivars when contrasted to their susceptible fruit peel ages. An exception occurred with CCR which was not enriched in the contrast of 14 to 10 dpp in ‘Chieftain’. 94 A B C C F 2 g o L 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 C14vsC7 C21vsC7 C14vsC10 C21vsC10 D21vsD7 Chieftain D21vsD10 Dickenson D21vsD14 Phenylalanine ammonia-lyase (4.3.1.24) (Cinnamate-4-hydroxylase) (Cytochrome P450 protein) Caffeoyl-CoA O-methyltransferase Figure 3.6.: Expression profile of squash orthologous genes that are differentially expressed (p<0.05, Log2(FC) >1, FC: Fold Change) in the different contrasts in both cultivars. A, B, C) DEGs involved in cutin, suberin monomers, and phenylpropanoid biosynthetic pathways respectively. 95 DISCUSSION We performed transcriptome profiling of the fruit peel of two C. moschata cultivars at developmental stages exhibiting susceptible and resistant phenotypes against P. capsici. K-means clustering showed two interesting groups of genes that were expected to include age-related resistance (ARR)-associated genes (Figure 3.1B, groups 1 and 2). To identify the candidate genes associated with ARR, we studied the gene expression profile between resistant and susceptible fruit peel ages in both cultivars. Comparison of gene expression profile along the cultivar time points and between cultivars resulted in consistently upregulated genes (Figure 3.2A) with enrichment in function for cell wall structures and phenylpropanoids biosynthesis (Figures 3.3, 3.4). Also, several of the downregulated genes detected in all comparisons between resistant and susceptible fruit peel ages were enriched in photosynthesis and cell growth (Figure 3.3). This was expected as fruit age increases and the fruit color of both cultivars changes from green to beige. Also, by 14 or 21 dpp in ‘Chieftain’ and 21 dpp in ‘Dickenson’, the fruits reaches their full expansion, and cell division is not likely to continue. While fruits are approaching complete development, other cellular processes including structural or metabolic may occur similar to cucumber (Ando et al. 2015, Mansfeld et al. 2017). Following the complete fruit expansion or elongation stage, cell wall structural changes occur with deposition of cell wall materials in the secondary cell wall such as lignin or xylan (Bacete et al. 2018). Deposition of primary cell wall polymers such as cellulose, hemicellulose, and pectin is less likely to occur since primary cell wall has been completed (Bacete et al. 2018). In different plant systems, ARR can be regulated by preformed or induced resistance mechanisms and both are regulated by changes in gene expression (Panter and Jones 2002, González-Lamothe 96 et al. 2009). Preformed defense mechanisms include physical barriers such as cell wall strengthening (Juge 2006) or chemical barriers such as the resistance of oat root against attack by the take-all disease of wheat (Osbourn et al. 1994). The plant cell wall constructs the physical barrier that all pathogens have to degrade to be able to infect and colonize the plant (Bellincampi et al. 2014, Bacetes et al. 2018). Changes in cell wall-related genes either by up or downregulation are significantly affecting disease resistance (Bellincampi et al. 2014, Meides et al. 2014). Among the upregulated genes detected in our study in all different comparisons within and between the two studied cultivars (Figure 3.2A, B, C), we identified a candidate group of ARR genes that function in lignin biosynthesis process, sinapyl alcohol dehydrogenase activity, cinnamyl alcohol dehydrogenase activity, stilbene biosynthetic process coumarin biosynthetic process, and peroxidase activity. These genes were upregulated consistently in resistant stages of both cultivars (Figure 3.3, Appendix Table 3. 2), which indicates that metabolic changes in the fruit cell wall are targeting the phenylpropanoids biosynthetic pathway since lignin, coumarin, stilbene, are products of the general phenylpropanoids pathway (Boerjan et al. 2003, Vogt 2010, Deng and Lu 2017). Genes encoding for peroxidase enzymes were expressed in all resistant stages in both cultivars (Figure 3.3, Appendix Table 3. 2). Peroxidases are oxidoreductases, cell wall heme-containing proteins (Vicuna 2005). They are involved in several physiological processes during plant development such as lignin polymerization, fruit ripening, and defense against biotic stress (Passardi et al. 2005). The last enzymatic step in lignin biosynthesis is catalyzed by peroxidases, which act to oxidize monolignols utilizing H2O2 (Higuchi 1985). Mutation in tomato has proofed the role of peroxidases in lignification (Quiroga et al. 2000). In addition to lignification, peroxidases are also involved in cell wall suberization (Quiroga et al. 2000). The biosynthesis and 97 deposition of suberin and lignin polymers in the plant secondary cell wall is developmentally regulated and provide strengthening to the cell wall to perform the physical barrier function against pathogen attack (Miedes et al. 2014, Pandey et al. 2017). Sinapyl alcohol dehydrogenase (SAD) and cinnamyl alcohol dehydrogenase (CAD) are enzymes in the phenylpropanoids pathway that catalyze the monolignols biosynthesis from phenylalanine (Vogt 2010, Miedes et al. 2014, Deng and Lu 2017). Multiple genes functionally annotated to be involved in lignin biosynthetic process were detected in the resistant stages of both ‘Chieftain’ and ‘Dickenson’, however, the highest fold change in the resistant fruit peel ages was for genes encoding for CCR and CAD (Figure 3.6C). In addition, genes encoding for ‘MYB transcription factors’ which are critical components of the lignin biosynthesis process were found significantly upregulated in several contrasts of both cultivars. A group of MYB proteins are demonstrated as a positive regulator of cell wall structure biosynthesis such as lignin (Zhou et al. 2009, Zhong et al. 2007, 2008) and cutin and consequently control cuticle and epidermis development (Oshima et al. 2013). Our findings suggest that ARR is potentially regulated in C. moschata winter squash cultivars through regulation of cell wall structures biosynthesis. We questioned which cell wall structure is the potential candidate that provides resistance to winter squash fruit against P. capsici. The pathway enrichment analysis detected enrichment in cutin, suberin monomers, and phenylpropanoids biosynthesis pathways in at least one resistant stage in both cultivars (Figure 3.4). However, enrichment in phenylpropanoids biosynthesis pathway was consistently detected in upregulated genes in all resistant stages in both cultivars (Figure 3.4). Therefore, we have examined the expression profile of individual genes involved in the three pathways. 98 In cutin biosynthesis, thioesterases are essential proteins for the release of de novo free fatty acids required for the biosynthesis process (Lowe 2010). The released fatty acids are in turn attached to CoA by the action of long chain acyl–CoA synthetases (LACs) (Schnurr et al. 2002). LACs enzymes are plant's long-chain fatty acid, AMP-dependent synthetase and ligase family protein (Li et al. 2016). The activation of acyl chains to acyl-CoAs by the LACs is an essential step in the biosynthesis of long chain fatty acids with variable lengths, which are required for cutin and cuticular wax biosynthesis. Cutin and cuticular wax are the components of plant cuticle, and provide a hydrophobic state to the outer plant surface, the cuticle, which acts as a protective barrier against abiotic and biotic stresses (Yeat and Rose 2013). In our results, the upregulated genes in ‘Chieftain’ and ‘Dickenson’ were enriched for multiple Acyl COA thioesterases and long-chain fatty acid CoA ligases that are required for the initial steps of cutin biosynthesis (Figure 3.6A). Genes encoding for thioesterase were upregulated in resistant 21 dpp in contrast to susceptible 7, 10, and 14 dpp in ‘Dickenson’ and in 14 and 21 dpp in contrast to 7 and 10 dpp in ‘Chieftain’ (Figure 3.6A). LACs gene was only upregulated in 21 dpp in contrast to 7 dpp in ‘Dickenson’ (Figure 3.6A), and was upregulated in resistant 14 and 21 dpp in ‘Chieftain’ in contrast to 7 and 10 dpp except the 14 vs.7 dpp was not differentially expressed (Figure 3.6A). LACs perform a key step in cutin and cuticular wax biosynthesis, and absence of their upregulation in ‘Dickenson’ at 21 dpp, when compared to susceptible 10 and 14 dpp (Figure 3.6A), might indicate that cutin biosynthesis in ‘Dickenson’ is not the potential mechanism for ARR as the fruit age increases. Phenylpropanoids are secondary metabolites that include flavonoids, lignin, coumarins, suberin and other phenolic compounds (Mander and Liu 2010). The phenylpropanoids biosynthesis pathway is derived from the shikimate pathway and begins with the amino acid phenylalanine (Mander and Liu 2010, Deng and Lu 2017). By the action of PAL, phenylalanine is converted to 99 cinnamic acid, then converted by C4H to p-coumaric acid then converted by 4CL to p-coumaroyl CoA, which is the precursor for several secondary metabolites including monolignols, stilbenes, coumarins, flavonoids and other phenolic compounds (Vogt 2010; Liu et al., 2015, Mander and Liu 2010). The monolignol biosynthesis has two routes, one of them is the conversion of p- coumaroyl-CoA to p-courmaraldhyde by CCR, then by the action of CAD, p-courmaraldhyde is converted to p-coumaryl alcohol that is used in building H lignin (Rinaldi et al., 2016). Another route starts by p-coumaric acid to produce G lignin, and S lignin utilizes the enzymes C4H and C3H to convert p-coumaric acid into caffeic acid then, with the action of another enzyme, it is turned into caffeoyl CoA. Caffeoyl CoA is the substrate for cCoAOMT to produce feruloyl CoA. Feruloyl CoA is then converted to coniferaldhyde by the catalysis of CCR. Coniferaldhyde is the substrate for CAD to produce the coniferyl alcohol, which is the building block for G lignin, or in another route, CAD/SAD convert the coniferaldhyde to sinapyl alcohol to end with building the S lignin (Deng and Lu 2017). The polymerization of the produced monolignols into lignin is catalyzed by peroxidases (Zhao et al. 2013, Rinaldi et al., 2016). Lignin is insoluble hydrophobic polymer forms a structural component of the secondary cell wall in plants (Tommerup and Andrews 1997, Miedes et al. 2014). Lignin provides structural support to the plant cell wall and has multiple functions including being a structural/physical barrier that defends against wounding and pathogens attack (Buendgen et al., 1990; Bonello et al., 2003, Labeeuw et al., 2015). Cell wall lignification has been known as a disease resistance mechanism in plants (Vance 1980, Nicholson and Hammerschmidt 1992, Sattler and Funnell-Harris 2013). The mechanical strength provided to the plant cell wall by lignin hinders the pathogen penetration using appressoria (Bellincampi et al. 2014). Also, the hydrophobic nature of lignin protects against cell wall degradation by the action of the cell wall degrading enzymes produced by plant pathogens (Vance 1980). cCoAOMT and 100 C4H genes were not upregulated in ‘Chieftain’ fruit peel of resistant ages 14 and 21 dpp when contrasted to susceptible 7 and 10 dpp (Figure 3.6B). Also, the two genes as well as PAL did not show significant changes in the contrast between 21 and 14 dpp in ‘Dickenson’ but were significantly upregulated in the resistant stage at 21 dpp vs. the 7 and 10 dpp stages (Figure 3.6B). This might indicate that the observed cell wall thickening in ‘Chieftain’ resistant fruit cuticle and epidermal walls (Alzohairy et al. 2017) that is correlated with ARR might not because of deposition of suberin and that another compound is causing this thickening, which is potentially the primary cause of ARR in hard squash to P. capsici. CAD, and CCR genes were enriched in the phenylpropanoid biosynthesis pathway and their expression was consistently upregulated in all the resistant fruit peel ages in both cultivars when contrasted to their susceptible fruit peel ages. However, CCR that did not change in the contrast of 14 vs.10 dpp in ‘Chieftain’ (Figure 3.6C). CAD and CCR are the primary two enzymes in the production of the monolignol p-coumaryl alcohol that is incorporated in building the H lignin (Rinaldi et al. 2016). In a study of the nature of lignin produced in cucumber hypocotyls, it was found that the lignin is derived from p-coumaryl alcohol (Robertsen and Svalheim 1990). The results suggest that CAD and CCR genes are potential candidate genes for controlling ARR through the production of monolignols and then polymerization of lignin at the cell wall by the catalysis of peroxidases. This additional lignin deposited in the peel of the resistant aging squash fruit may provide the thickening to the cuticle and the epidermal walls to resist against pathogens and environmental stresses. Also, the observed increasing of cCoAOMT and C4H expression in the resistant fruit are likely involved in the production of lignin and not suberin. 101 CONCLUSION Our results provide evidence that the ARR to P. capsici in winter squash is highly correlated with phenylpropanoids production and that lignin is the potential material that is deposited in the cuticle and epidermal cell walls of maturing fruit peel as a constitutive structural defense mechanism against pathogens. Although the ARR onset in the tested cultivars was different, several genes detected in both cultivars were consistently upregulated in the resistant fruit peel and were enriched in phenylpropanoids biosynthesis. This suggests that both C. moschata cultivars have similar mechanisms that control ARR to P. capsici. The observed difference in the onset of ARR between cultivars is likely due to the difference in their days to maturity as ‘Chieftain’ matures at 80 days while ‘Dickenson’ matures at 100 days. 102 APPENDIX 103 APPENDIX Table 3.1.: Number of raw reads, alignment count, and uniquely mapped reads. Sample No. C7-1 C7-2 C7-3 C10-1 C10-2 C10-3 C14-1 C14-2 C14-3 C21-1 C21-2 C21-3 D7-1 D7-2 D7-3 D10-1 D10-2 D10-3 D14-1 D14-2 D14-3 D21-1 D21-2 D21-3 Number of sequenced reads QC-pass and mapping input Uniquely mapped Mapping % 55113437 66386572 56539714 44396147 51954298 59773579 175601685 58883472 168147746 63093646 69714639 55535461 43697048 39042901 41852230 40997570 52014876 47521845 54894218 43914637 47600991 51963759 49840126 44596852 50288035 60788885 51567392 40454480 46255164 53981968 162531776 53892199 154877625 56908664 63134472 48999524 38574146 35207320 38426892 37564434 46467810 42776661 49579501 39735017 42138426 46334848 44627977 39845052 63739767 70456945 59026276 44371192 60930638 63003314 193,631,816 65821663 180,842,987 67405839 70485362 57362566 48126734 42862633 45528798 44350665 58118081 54015612 59724520 49203062 54435378 56384199 55199091 49742926 91.24 91.57 91.21 91.12 89.03 90.31 92.56 91.52 92.11 90.2 90.56 88.23 88.28 90.18 91.82 91.63 89.34 90.01 90.32 90.48 88.52 89.17 89.54 89.34 104 Table 3.2.: Gene Ontology enrichment of upregulated genes in the resistant stages in both cultivars resulted from all contrasts. The values are the negative log of the q-value if the GO term is overrepresented, or the log of the q-value if the GO term is underrepresented. At q = 0.05, a term is significantly overrepresented if the value is > or = to 1.3 and significantly underrepresented if the value is < or = to -1.3. C21 up_ vs_ C7 C21 up_ vs_ C10 D21 up_v s_D 7 D21 up_ vs_ D10 D21 up_ vs_ D14 Set 3 GO-term GO:0003677_DNA_binding GO:0005634_nucleus GO:0055114_oxidation- reduction_process GO:0008270_zinc_ion_binding GO:0005515_protein_binding GO:0006687_glycosphingolipid _metabolic_process GO:0046486_glycerolipid_meta bolic_process GO:0000785_chromatin GO:0003682_chromatin_bindin g GO:0046872_metal_ion_bindin g GO:0005829_cytosol GO:0004565_beta- galactosidase_activity GO:0009341_beta- galactosidase_complex GO:0006027_glycosaminoglyca n_catabolic_process GO:0005622_intracellular GO:0006012_galactose_metabol ic_process Set 1_u p Set2 _up_ Chie ftain 3.3 21 3.3 21 2.6 37 2.6 37 2.6 37 1.9 64 1.9 64 1.9 64 1.9 64 1.9 64 1.9 64 1.3 1.3 1.3 1.3 1.3 0.97 2 - 0.11 8 3.82 4 0 - 1.45 8 2.14 7 1.16 8 1.16 1 1.09 1 0.28 3 0.15 1 1.80 2 1.80 2 1.80 2 1.28 4 1.09 9 Set2 _up _Di cken son 2.43 3 0 2.38 8 1.89 7 - 0.90 8 1.78 8 0.87 9 2.15 2 2.26 4 0.47 5 - 0.94 4 1.36 1 1.36 1 1.36 1 0.59 0.15 9 C14 up_ vs_ C10 4.37 C14 up_ vs_ C7 0.30 2 0 0.52 9 1.60 3 - 0.29 8 - 2.19 8 0.70 6 0.24 7 2.24 7 - 1.37 3 - 4.33 3 0.16 7 - 0.50 4 0 0.84 1 0.59 8 - 0.22 8 0.36 1.56 4 - - 2.87 0.70 9 1 0 0.64 8 0 0.64 8 0 0.64 8 0.16 1 - 0.14 3 0.43 1.02 5 105 1.96 5 - 2.00 4 2.89 5 0.21 4 - 4.27 5 0.14 3 - 0.25 5 1.97 2.02 3 0.11 3 - 5.34 6 0.18 4 0.18 4 0.18 4 0 - 0.30 9 - 0.57 9 3.00 1 - 2.93 1 - 3.37 2 0.57 1 - 0.25 8 - 0.68 - 1.06 8 0.19 5 - 0.70 1 0.27 4 0.27 4 0.27 4 - 0.15 4 0.47 1 1.0 76 - 0.3 79 0.9 56 - 0.9 43 0.2 76 0.6 34 0 - 0.4 97 - 0.7 13 0 - 2.9 09 0.2 97 0.2 97 0.2 97 - 0.4 03 0.5 11 0.42 5 3.86 4 3.07 5 0 3.45 9 2.49 2 1.54 9 6.07 0 0 1.08 4 1.04 3 - 2.12 3 1.15 4 0.41 3 - 0.32 5 2.12 5 - 0.54 5 - 10.8 1 0.15 8 - 0.39 9 0.48 2.75 0.39 4 0.49 5 2.88 2 0.40 2 0.40 0.53 6 2 - - 2.04 2.18 7 3 0 0.92 4 0 0.92 4 0 0.92 4 0 0.77 4 1.74 1.36 3 0 0 0 0 0 - 0.38 6 - 1.31 1 - 0.12 7 Table 3. 2 (cont’d) GO:0030246_carbohydrate_bin ding 1.3 0.57 5 0.5 - 0.38 0 - 2.21 2 - 3.57 7 2.36 0 1.74 4.63 5 0.21 1 0.41 1 0.41 1 0 0 0 0 0.21 1 0 0 0 0 0.40 2 0 0 0 0 0 0.5 88 - 3.0 15 0.1 77 1.7 26 1.0 58 0.3 36 0.2 97 0.2 97 1.1 64 0.2 97 0.8 84 0.2 78 1.0 92 0.8 61 1.3 25 0.8 0.3 29 - 0.3 19 - 0.2 28 0 0 0.89 7 - 3.81 6 - 4.88 8 0.44 6 2.99 4 2.00 7 0.54 0.54 0.40 6 0.78 7 0.12 1.69 4 0.59 5 0.59 5 0 2.23 5 1.57 3 0.56 5 0.58 5 0.38 1 0.47 6 - 0.09 9 - 1.61 5 - 2.61 2 3.47 8 3.66 3 1.85 6 0.52 6 0.52 6 1.29 6 0.27 4 1.55 1 1.41 4 0.99 2 0.77 8 0.57 1 0.35 7 0.64 3 - 0.28 8 0 - 0.19 9 - 0.45 8 0.82 9 - 0.61 7 - 1.39 9 - 10.9 1 0.39 9 2.98 1 0.66 3 0.59 1 0.59 1 0.61 1 0.29 7 - 0.13 7 1.52 3 1.12 8 - 0.81 2 - 0.70 5 0.19 6 1.11 7 0.53 3 1.33 4 1.33 4 0.92 7 0.66 7 - 0.40 5 0.77 1 0.97 1 0.8 0.41 9 0.57 6 2.73 8 1.33 4 0.09 1 1.67 5 2.01 3 0.27 8 0.63 7 0.86 6 0.70 7 0.92 7 0.92 4 0.92 4 2.18 8 GO:0009507_chloroplast 1.3 -0.16 GO:0005524_ATP_binding GO:0005506_iron_ion_binding GO:0020037_heme_binding GO:0016491_oxidoreductase_ac tivity GO:0052747_sinapyl_alcohol_d ehydrogenase_activity GO:0045551_cinnamyl- alcohol_dehydrogenase_activity GO:0009811_stilbene_biosynth etic_process GO:0042753_positive_regulatio n_of_circadian_rhythm GO:0005618_cell_wall GO:0006804_peroxidase_reacti on GO:0009805_coumarin_biosynt hetic_process GO:0009809_lignin_biosyntheti c_process GO:0030001_metal_ion_transpo rt GO:0046983_protein_dimerizati on_activity GO:0019852_L- ascorbic_acid_metabolic_proces s GO:0004712_protein_serine/thr eonine/tyrosine_kinase_activity GO:0005887_integral_compone nt_of_plasma_membrane GO:0008378_galactosyltransfer ase_activity GO:0009117_nucleotide_metab olic_process - 0.73 5 4.78 5 4.19 7 2.37 9 1.80 2 1.80 2 1.80 2 1.80 2 1.56 1.53 2 1.36 3 1.36 3 1.36 3 1.1 1.09 9 0.66 4 0.47 8 0.47 8 0 1.3 0.6 46 0.6 46 0.6 46 0.6 46 0.6 46 0.6 46 0.6 46 0.6 46 0.6 46 0.6 46 0.6 46 0.6 46 0.6 46 0.6 46 0.6 46 0.6 46 0.6 46 0.6 46 - 0.50 6 - 0.85 1 0.89 2 1.83 0.31 2 1.80 1 1.80 1 1.52 9 0.9 0 1.77 8 1.73 2 1.16 5 0 2.73 6 1.80 1 1.52 9 1.36 1 1.36 1 1.78 8 - 1.06 4 - 10.6 7 2.30 7 3.44 5 0.85 5 2.29 3 0.60 7 0.25 0.64 8 0.25 0.64 8 0.97 0.71 7 2 0 0.64 8 0.86 1.34 2 1.77 8 0.52 4 0.34 4 1.47 4 0.40 7 0.13 1 0.88 7 0.70 6 0.70 6 3.52 9 1.29 5 0.17 2 - - 0.13 0.79 1 7 0 0.17 2 - 0.20 2 - 1.69 9 0 - 0.29 1 106 Table 3. 2 (cont’d) GO:0006468_protein_phosphor ylation 0.6 46 0 GO:0042742_defense_response _to_bacterium 0 1.80 2 GO:0006118_electron_transport 0 1.56 - 1.28 9 - 0.21 2 0 - 3.68 3 1.83 5 - 0.87 8 2.59 8 2.95 3 0.35 8 GO:0004601_peroxidase_activit y GO:0042744_hydrogen_peroxid e_catabolic_process GO:0009055_electron_carrier_a ctivity GO:0010167_response_to_nitrat e GO:0010089_xylem_developme nt GO:0015706_nitrate_transport GO:0009723_response_to_ethyl ene GO:0048437_floral_organ_deve lopment GO:0000150_recombinase_acti vity GO:0045489_pectin_biosyntheti c_process GO:0044237_cellular_metabolic _process GO:0004715_non- membrane_spanning_protein_ty rosine_kinase_activity GO:0043565_sequence- specific_DNA_binding GO:0018108_peptidyl- tyrosine_phosphorylation GO:0009743_response_to_carb ohydrate GO:0045449_regulation_of_tran scription GO:0005667_transcription_fact or_complex GO:0003700_sequence- specific_DNA_binding_transcri ption_factor_activity 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1.36 3 1.36 3 1.36 3 0.9 0.9 0.47 8 0.47 8 0 0 0 0 0 0 0 0 - 0.11 9 - 0.11 9 -0.12 1.40 3 1.32 8 0.17 1 1.80 1 1.36 1 1.78 8 1.36 1 1.80 1 1.80 1 1.80 1 1.78 8 1.73 2 1.43 5 1.38 5 1.36 1 1.02 8 0.65 5 1.04 9 1.87 1 1.26 1 4.09 9 1.51 1 0.96 7 1.51 1 2.03 0.64 8 0 0.97 2 0 0.75 0.43 0.36 5 0 0.21 7 0 0 - 1.62 9 0.34 4 - 0.94 1 0.99 1 - 1.63 4 0 1.43 8 1.63 6 1.57 2 - 0.20 3 0.70 6 0 1.20 3 - 0.09 3 0 5.88 9 5.13 3 5.63 4 107 - 0.72 6 2.03 8 2.39 2 1.41 3 1.41 3 3.20 9 2.29 2 0.51 7 1.01 5 0.47 1 - 0.42 8 0 - 0.82 5 0.18 3 - 0.45 8 1.45 7 - 1.29 8 0 1.17 7 1.48 4 1.41 3 1.3 39 2.8 18 0.8 84 0.6 34 0.6 34 1.5 47 1.1 64 1.4 46 0.5 11 0.7 02 0 0 0 1.0 92 0.0 98 3.0 23 0 0 6.0 31 5.6 84 5.4 48 - 0.19 5 - 0.31 6 - 0.15 2 1.17 5 1.47 4 0.06 9 3.55 1 0 2.02 6 0.99 9 0.54 0.54 0.78 6 0.14 3 0.45 9 3.23 3 0.14 4 0 2.89 1 2.08 9 2.82 3 0.27 3 - 4.86 7 - 0.09 - 0.54 7 - 0.15 5 - 0.55 3 0.57 6 0.88 0.75 6 0 0.97 5 0.78 1 3.91 8 2.67 1 0.2 1.47 6 2.18 8 0.92 4 1.33 4 2.01 1 1.09 9 0.29 7 0.29 7 0.59 1 0.51 5 0.22 6 2.00 2 2.00 2 1.15 4 1.35 9 3.19 3 0.63 9 1.56 4 - 0.28 4 0 1.47 6 3.64 3 0.68 5 1.72 3 0 0.41 1 0 0 0 0 0 0 0 0 0 0 0 0 0.58 0 0 1.36 3 0.47 1 0.62 5 2.29 8 1.35 8 3.47 1 1.15 2 0 2.29 7 - 0.32 5 - 0.08 7 - 0.43 1.54 9 1.36 3 1.36 3 0.48 2 2.11 9 4.04 3 - 0.13 8 -2.6 1.36 3 Table 3. 2 (cont’d) GO:0008152_metabolic_process 0 -0.15 0 GO:0009069_serine_family_am ino_acid_metabolic_process GO: 0003735_structural_constituent_ of_ribosome GO:0042254_ribosome_biogene sis GO:0005886_plasma_membran e GO:0016021_integral_compone nt_of_membrane GO:0005739_mitochondrion 0 0 0 0 0 0 - 0.34 2 - 0.11 9 - 0.24 6 0.17 7 - 0.18 8 - 1.41 - 0.19 1 - 0.20 5 - 0.20 5 - 0.25 1 - 1.01 2 - 1.11 4 - 2.60 3 - 3.26 5 21.8 74 23.5 76 - 0.25 3 - 4.55 2 0 - 0.81 5 - 1.06 3 19.2 75 20.9 04 - 0.43 8 - 1.54 6 1.00 4 - 1.82 4 - 0.59 1 3.42 9 3.84 4 - 0.68 - 3.84 4 - 1.26 1 - 0.8 11 0.6 57 - 0.1 91 - 0.3 22 - 0.4 77 - 0.4 56 - 0.1 2 - 1.32 7 - 2.81 1 20.7 21 19.8 96 - 0.32 3 - 1.15 8 0.29 7 - 0.27 0.06 1 - 1.23 7 - 1.26 8 - 0.33 9 - 1.01 3 - 0.57 7 108 Table 3.3.: Gene Ontology enrichment of downregulated genes in the resistant stages in both cultivars resulted from all contrasts. The values are the negative log of the q-value if the GO term is overrepresented, or the log of the q-value if the GO term is underrepresented. At q = 0.05, a term is significantly overrepresented if the value is > or = to 1.3 and significantly underrepresented if the value is < or = to -1.3. GO-term GO:0016021_integral_compon ent_of_membrane GO:0009535_chloroplast_thyla koid_membrane GO:0055114_oxidation- reduction_process GO:0046872_metal_ion_bindin g GO:0016168_chlorophyll_bindi ng GO:0019344_cysteine_biosynt hetic_process GO:0006364_rRNA_processin g GO:0005515_protein_binding GO:0018298_protein- chromophore_linkage GO:0009522_photosystem_I GO:0009941_chloroplast_envel ope GO:0009570_chloroplast_stro ma GO:0005524_ATP_binding GO:0009765_photosynthesis GO:0006098_pentose- phosphate_shunt GO:0009523_photosystem_II GO:0019288_isopentenyl_diph osphate_biosynthetic_process Set1 _do wn 10.2 69 5.05 7 5.05 7 4.72 7 4.07 3 4.07 3 4.07 3 3.74 8 3.42 6 3.42 6 3.42 6 3.42 6 3.42 6 Set2 _do wn_ Chi eftai n 7.32 3 7.34 7 4.51 5 2.04 9 9.57 5 6.21 2 5.33 1 - 0.98 9 7.82 8 7.16 9 4.96 1 1.98 6 0.11 8 3.10 5 3.10 5 2.78 7 7.50 3 6.35 5 6.81 5 2.47 2.85 2 Set2 _do wn_ Dic ken son 6.43 2 23.4 01 5.60 7 1.09 5 13.2 02 8.82 6 11.4 69 - 3.47 7.15 4 14.0 18 6.60 8 2.52 5 - 1.50 7 10.9 89 7.14 8 13.2 02 4.69 5 C14 dow n_v s_C 7 C14 dow n_v s_C 10 C21 dow n_v s_C 7 C21 dow n_v s_C 10 D21 dow n_v s_D 7 D21 dow n_v s_D 10 D21 dow n_v s_D 14 2.95 2 0.12 7 1.10 2 0.38 9 1.90 3 0.07 5 - 0.06 6 2.15 7 1.73 7 0.84 2 0.05 4 - 1.84 7 0.76 8 1.51 9 0.14 8 1.72 8 - 0.36 5 8.41 6 6.10 5 5.25 5 1.70 8 5.99 5 2.96 4 5.28 5 - 0.79 3 4.46 3.91 4 4.41 3 1.55 3 - 0.12 6 4.32 3 6.60 9 5.16 1 2.72 9 3.81 6 3.19 8 2.16 5 0.80 8 2.94 9 1.31 9 0.85 7 - 0.73 1 2.05 1 2.05 1 0.66 7 - 0.97 2 - 0.68 4 2.02 8 1.64 7 2.94 9 0.21 7 4 13.2 87 4.69 8 1.63 2 5.61 8 3.38 7 5.83 9 - 3.14 1 4.24 9 4.48 7 6.04 9 1.22 - 4.13 7 3.83 9.23 8 6.41 5 2.78 2.28 8 10.0 63 5.18 9 0.26 2 4.96 1.88 5 5.05 9 - 6.64 2 1.40 3 5.61 5 3.61 1.11 - 1.17 3.39 8 1.81 9 4.96 4.74 5 2.26 8 15.5 31 3.79 0.23 9 6.46 3 3.07 6 7.75 - 3.81 8 2.3 7.19 6.74 8 3.70 1 - 0.72 3 4.35 4 4.14 7 6.46 3 7.97 2 5.55 3 18.0 91 5.23 4 0.47 9 10.2 91 6.80 5 8.47 7 - 4.44 7 5.15 8 11.1 56 5.52 2 1.71 5 - 0.96 8 8.75 1 5.50 3 10.2 91 4.02 8 Set 3 4.04 3 0.41 1 2.49 2 1.74 0 0 0 6.07 0 0 0.21 2 0.39 4 4.63 5 0 0 0 0 109 Table 3. 3 (cont’d) GO:0010207_photosystem_II_a ssembly GO:0009637_response_to_blue _light GO:0019252_starch_biosynthet ic_process GO:0010218_response_to_far_ red_light GO:0035304_regulation_of_pr otein_dephosphorylation GO:0010155_regulation_of_pr oton_transport GO:0010287_plastoglobule GO:0009507_chloroplast GO:0005634_nucleus GO:0010103_stomatal_comple x_morphogenesis GO:0048046_apoplast GO:0010114_response_to_red_ light GO:0015995_chlorophyll_bios ynthetic_process GO:0010027_thylakoid_membr ane_organization GO:0006096_glycolytic_proces s GO:0009644_response_to_high _light_intensity GO:0005985_sucrose_metaboli c_process GO:0003677_DNA_binding GO:0005886_plasma_membran e GO:0015976_carbon_utilizatio n GO:0009773_photosynthetic_el ectron_transport_in_photosyste m_I GO:0071555_cell_wall_organiz ation 2.15 5 2.15 5 1.84 2 1.84 2 1.84 2 1.84 2 1.84 2 1.84 2 1.84 2 1.53 1 1.53 1 1.53 1 1.53 1 1.53 1 1.53 1 1.53 1 1.53 1 1.53 1 1.53 1 1.22 1 1.22 1 1.22 1 6.41 4 5.17 4 4.85 3 4.57 6 3.96 5 3.48 8 3.37 5 0.51 1 - 2.52 6 6.12 8 5.86 9 2.79 7 2.58 4 2.04 3 1.25 1 0.91 5 0.29 6 0 - 0.26 4 3.94 1 3.39 3 3.26 9 12.3 55 9.29 8 3.16 2 7.92 8 5.11 2 5.15 4 6.75 5 - 0.03 7 - 3.19 3 4.31 3 6.09 2 7.85 3 7.00 2 2.90 5 0.19 4 5.31 1 10.0 23 0.54 3 0 2.00 4 6.42 2 2.39 7 1.35 8 0.55 7 0.39 4 0.84 2 0.55 0.35 3 0.12 8 - 1.85 - 1.16 4 2.96 7 2.96 2 0.26 5 0.2 - 0.15 1 - 0.07 4 - 0.52 4 1.53 6 0.21 4 0.30 1 0.84 2 0.44 1 2.30 4 8.14 4 4.51 7 3.54 5 4.46 3.75 3 2.71 4 2.26 4 1.94 8 - 3.21 6 5.16 1 4.03 9 3.24 9 3.13 9 2.72 9 1.96 9 0.36 0.10 8 - 1.31 0 4.18 7 4.32 3 0.98 3.72 1 2.36 2 0.66 3 2.67 9 2.18 1 0.96 4 0.92 2 - 1.96 2 - 1.39 3 4.92 5 4.99 1 1.44 6 1.46 5 0.13 9 0.14 7 - 0.27 1.63 8 1.00 8 0.45 1 1.67 9 2.27 8 3.13 7 10.3 99 6.33 5 4.19 7 6.38 3 4.48 9 3.06 1 2.70 2 0.79 7 - 1.31 6 5.89 2 5.10 5 4.93 7 5.32 6 2.62 1.06 2 0.07 2 0.41 9 0.42 8 - 0.33 2 4.48 7 5.61 8 1.88 1 7.33 9 5.37 6 0.79 3.93 3 2.03 6 0.73 8 1.45 2 0.02 5 - 1.78 6 3.93 3 2.43 4 3.93 2 7.13 8 2.87 2 - 1.88 4 1.47 8 5.80 2 2.84 1 - 0.20 9 0 3.51 8 1.14 3 10.0 25 6.88 8 2.29 6 5.25 3 2.83 2 1.81 4 2.28 6 0.52 1 - 3.87 6 5.49 2 3.34 2 5.31 9.16 3 5.44 2 - 1.01 2 2.64 1 6.23 9 0.93 7 - 0.53 9 0.64 7 4.64 7 0.66 10.6 17 7.97 9 3.01 7 6.36 2 4.04 3 3.47 6 5.32 3 0.06 8 - 3.30 3 4.22 8 6.88 3 6.45 7 5.57 3 1.94 7 0.10 8 5.87 6.54 6 0.57 7 - 0.02 9 1.84 8 6.07 9 2.35 3 0 0 0 0 0 0 0 1.74 3.45 9 0 0 0 0 0 0 0 0.41 1 3.07 5 2.11 9 0 0 0 110 Table 3. 3 (cont’d) GO:0009744_response_to_sucr ose GO:0019684_photosynthesis GO:0051287_NAD_binding GO:0005215_transporter_activi ty GO:0006810_transport GO:0008152_metabolic_proces s GO:0005982_starch_metabolic _process GO:0009543_chloroplast_thyla koid_lumen GO:0000023_maltose_metaboli c_process GO:0043085_positive_regulati on_of_catalytic_activity GO:0070838_divalent_metal_i on_transport GO:0030003_cellular_cation_h omeostasis GO:0010196_nonphotochemica l_quenching GO:0009902_chloroplast_reloc ation GO:0009657_plastid_organizati on GO:0004650_polygalacturonas e_activity GO:0005739_mitochondrion GO:0008270_zinc_ion_binding GO:0005506_iron_ion_binding GO:0020037_heme_binding GO:0006855_drug_transmembr ane_transport GO:0006118_electron_transpor t GO:0019253_reductive_pentos e-phosphate_cycle 1.22 1 1.22 1 1.22 1 1.22 1 1.22 1 1.22 1 1.22 1 0.91 3 0.91 3 0.91 3 0.91 3 0.91 3 0.91 3 0.91 3 0.91 3 0.91 3 0.91 3 0.91 3 0.60 7 0.60 7 0.60 7 0.60 7 0.60 7 2.69 7 2.23 5 2.09 1.63 4 1.38 6 0.24 3 0.18 8 3.39 3 3.26 9 2.77 2.71 2 2.71 2 2.71 2 2.29 2.09 2 2.09 2 - 0.30 1 - 1.79 3.44 7 2.88 7 2.09 2 2.03 9 2.03 2 4.36 8 1.87 3 2.92 6 0.18 0.18 - 1.00 9 9.25 3 4.54 2 2.92 6 4.09 2 5.19 6 4.59 4 2.27 3 2.04 7 9.48 2.48 2 - 2.19 3 - 1.77 3 1.28 1 2.11 4 1.46 6 0.69 7 3.62 1.55 7 0 0 0 - 0.06 4 0.73 5 1.76 6 0.67 5 0.08 8 0 0.65 0.88 7 0.22 6 0 0.67 0.49 6 - 3.77 6 - 0.14 9 2.44 6 0.82 5 1.37 7 0.69 0.45 9 3.38 8 1.36 1.65 1 0.79 0.55 8 0.41 2 0.11 4.32 3 2.29 5 2.09 5 3.48 3.48 1.18 5 2.49 7 3.64 1 0.64 8 - 0.48 - 1.14 7 4.21 1 1.89 5 2.87 7 1.76 3 2.34 3 2.48 0.13 1 0 0.75 7 0.20 7 - 0.24 2.10 1 1.58 6 0.19 3 0.19 3 2.50 7 2.46 7 0.23 4 0.79 4 1.47 4 0.33 6 - 4.72 4 - 2.75 4 2.47 1 0.87 5 1.47 5 2.30 7 1.17 1 3.06 5 1.64 1 0.51 1.41 1 0.61 7 - 0.54 0.28 7 5.38 2 3.00 1 2.61 9 4.89 2 5.14 1.05 8 1.88 1 4.48 7 0.59 - 1.41 2 - 1.24 1 3.57 7 1.70 9 1.00 7 3.95 4 2.85 7 0.80 5 0.49 7 0.30 2 - 0.05 7 - 0.05 8 - 0.72 7 6.67 2 1.86 5 1.35 4 1.62 7 1.81 6 1.45 2 0.16 7 2.63 3 4.70 6 1.84 7 - 2.76 1 - 5.20 9 1.15 2 1.67 3 0.96 8 0.97 8 0.90 2 1.40 8 0.91 7 0.31 7 - 0.21 - 0.05 9 - 0.16 6.67 3.09 3 2.98 9 3.19 8 2.95 3 2.48 5 0.36 3 4.30 5 6.46 5 1.49 6 - 1.65 8 - 4.15 2 0.75 3 1.08 3 0.69 6 0.95 3 2.01 2 2.78 8 1.32 3 2.14 4 0.25 0.69 5 - 0.57 7 6.34 2 3.48 1 2.66 8 3.74 5 4.57 7 3.79 8 1.71 4 1.43 1 8.35 1 1.97 9 - 1.95 2 - 1.58 6 1.07 1 1.73 5 0.99 6 0.89 9 3.48 1 0 0 0 0 0 2.29 7 0.41 1 0 0 0 0 0 0 0 0 0 1.36 3 1.54 9 0.21 1 0.41 1 0 0.41 1 0 111 Table 3. 3 (cont’d) GO:0009783_photosystem_II_a ntenna_complex GO:0005576_extracellular_regi on GO:0009409_response_to_cold GO:0050661_NADP_binding GO:0051537_2_iron GO:0006804_peroxidase_reacti on GO:0019852_L- ascorbic_acid_metabolic_proce ss GO:0019761_glucosinolate_bio synthetic_process GO:0050794_regulation_of_cel lular_process GO:0006468_protein_phosphor ylation GO:0006633_fatty_acid_biosyn thetic_process GO:0009055_electron_carrier_ activity GO:0010075_regulation_of_me ristem_growth GO:0043481_anthocyanin_acc umulation_in_tissues_in_respo nse_to_UV_light GO:0016705_oxidoreductase_a ctivity GO:0032440_2- alkenal_reductase_[NADP]_act ivity GO:0009595_detection_of_biot ic_stimulus GO:0043900_regulation_of_mu lti-organism_process GO:0010310_regulation_of_hy drogen_peroxide_metabolic_pr ocess GO:0042132_fructose_1 GO:0008356_asymmetric_cell_ division GO:0019760_glucosinolate_me tabolic_process 0.60 7 0.60 7 0.60 7 0.60 7 0.60 7 0.60 7 0.60 7 0.60 7 0.60 7 0.60 7 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 2.03 2 1.65 8 1.50 4 1.50 4 1.50 4 1.40 3 1.17 9 0.95 0.95 - 0.20 8 2.77 2.69 7 1.69 3 1.69 3 1.62 1.42 7 1.35 3 1.35 3 1.35 3 1.35 3 1.35 3 1.35 3 1.48 8 7.12 6 1.09 7 0.93 5 0.66 7 0.75 6 3.97 6 3.35 6 1.38 6 0.03 8 1.25 5 0.47 7 2.26 0.27 1.62 3 1.69 6 3.62 3.02 5 2.19 9 1.98 5 1.48 8 1.48 8 0 1.80 5 1.39 1 0.32 6 0.16 0.35 2 0.55 7 0.59 6 0.92 6 2.62 2 1.61 5 1.12 4 0.62 3 0.93 1 0 1.6 0.76 0.85 1 1.14 1.01 9 2.38 4 0.71 1 0.09 7 1.11 6 0.22 6 0.22 6 0.65 0 0.44 1 0 0.13 6 - 0.27 8 2.70 3 2.11 6 2.02 5 0.52 6 1.07 1 1.31 2 1.18 5 1.18 5 1.18 5 1.18 5 0.29 9 0.59 6 0.25 4.27 2.07 7 0 0.16 7 0.11 4 0.42 6 0.51 1 0.64 9 0.1 1.36 2.88 6 4.51 3 1.98 6 0.10 2 2.18 3 1.17 1 1.17 1 1.83 5 0.23 4 0.94 0.25 0.54 6 2.98 4 3.51 9 0.38 4 2.12 7 0.56 6 0.47 8 1.44 6 0.12 6 - 0.65 7 1.20 5 4.48 7 3.14 2 1.22 4 0.23 3 1.81 4 2.08 9 2.08 9 2.31 7 1.32 6 0.79 8 0.79 8 0.25 2 6.51 6 0.41 - 0.10 9 - 0.22 3 0.43 5 2.95 9 0.80 5 0.33 5 0.34 8 1.18 2 1.31 2.11 4 0.25 7 0.87 2 1.86 3 1.07 9 0.75 7 0.87 2 0.48 1 1.62 8 0.25 2 0.25 5 6.30 1 1.13 - 0.11 - 0.09 8 0.25 5 2.68 8 2.10 5 0.25 3 0.58 9 1.86 1.39 5 1.95 1 0 1.07 8 1.72 2.21 3 1.74 2 1.57 2 0.98 2 2.20 9 0.51 6 112 0 0.21 1 0 0 0 0 0 0 0 1.18 7 5.90 4 1.65 1 0.61 5 0.40 1 0.64 1 3.17 2.91 1.33 1 0.07 1.72 3 2.78 8 1.30 8 1.84 8 0 1.11 0 0 0 0 0 1.25 5 0.40 2 3.84 6 3.2 2.34 3 1.97 9 1.18 7 1.58 3 0 0 0 0 0 0 Table 3. 3 (cont’d) GO:0009517_PSII_associated_l ight-harvesting_complex_II GO:0010277_chlorophyllide_a _oxygenase_[overall]_activity GO:0050278_sedoheptulose- bisphosphatase_activity GO:0042908_xenobiotic_transp ort GO:0008559_xenobiotic- transporting_ATPase_activity GO:0006006_glucose_metaboli c_process GO:0016620_oxidoreductase_a ctivity GO:0009749_response_to_gluc ose GO:0015979_photosynthesis GO:0050832_defense_response _to_fungus GO:0010200_response_to_chiti n GO:0006566_threonine_metab olic_process GO:0046658_anchored_compo nent_of_plasma_membrane GO:0019216_regulation_of_lip id_metabolic_process GO:0006544_glycine_metaboli c_process GO:0010206_photosystem_II_r epair GO:0009862_systemic_acquire d_resistance GO:0008361_regulation_of_cel l_size GO:0009697_salicylic_acid_bi osynthetic_process GO:0010363_regulation_of_pla nt- type_hypersensitive_response GO:0009867_jasmonic_acid_m ediated_signaling_pathway GO:0006612_protein_targeting _to_membrane GO:0009416_response_to_light _stimulus GO:0006636_unsaturated_fatty _acid_biosynthetic_process 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 1.35 3 1.35 3 1.35 3 1.35 3 1.35 3 1.35 3 1.35 3 1.35 3 1.17 9 0.94 2 0.94 2 0.94 2 0.94 2 0.94 2 0.94 2 0.94 2 0.94 2 0.94 2 0.70 5 0.70 5 0.70 5 0.70 5 0.70 5 0.70 5 1.48 8 0.99 2 0.99 2 0.61 8 0.61 8 0.61 8 0.61 8 0.16 3 6.31 5 3.66 2 3.29 3.02 5 2.71 6 2.48 2 2.19 9 1.98 5 1.76 4 1.35 2.93 2.11 7 2.04 7 2.00 4 1.98 5 1.81 9 0 0 0 0.44 1 0.44 1 0 0 0.65 0 1.55 7 1.91 7 0.32 4 0.84 7 0 0.32 4 0 0.49 4 0.66 7 0 1.02 9 0.40 8 1.02 9 - 0.18 0 0.59 6 0.59 6 0.59 6 0.89 2 0.89 2 0.59 6 0.59 6 1.18 5 3.50 1 0.88 3 0.88 3 1.86 5 0.20 2 0.42 2.36 8 0.20 2 0.64 8 0.42 0.52 6 0.52 6 0.33 7 0.52 6 0.16 0.52 6 0 0 0 0.23 4 0.23 4 0 0 0.71 8 1.44 6 1.65 2 2.48 0.71 1.87 1 0 0.70 3 0.16 7 1.28 6 1.47 5 0.43 1 1.99 1 1.09 1 1.99 1 0 0.13 4 0.54 6 0.79 8 0.54 6 0.54 6 0.54 6 0.28 2 0.28 2 0.54 6 5.64 7 1.90 7 1.45 6 0.59 1.00 7 0 0.59 0.59 1.90 7 1.45 6 1.03 8 1.43 2 1.22 8 1.43 2 0.30 4 1.22 8 0.25 2 0.25 2 0 0.16 9 0.16 9 0 0 - 0.12 2 4.18 4 1.21 5 3.14 6 0.74 7 1.86 5 1.17 8 0.21 3 0.94 4 0.66 1 0.87 2 1.16 5 1.25 0.79 7 0.94 5 1.39 7 1.29 1 0.25 5 0.26 3 0.25 5 0.36 3 0.36 3 0 0 0 6.15 6 2.45 5 2.45 5 0.98 4 2.66 3 1.73 6 0.22 2 0.98 2 1.45 0.92 5 2.09 5 1.19 3 1.45 0.87 8 1.96 3 2.48 5 1.18 7 1.18 7 1.18 7 0.24 5 0.24 5 0.24 5 0.24 5 0 5.66 6 3.47 2 4.00 6 2.02 2.05 7 1.97 9 1.34 6 1.58 3 2.14 4 0.88 3 2.84 8 2.40 1 2.93 5 2.04 8 1.97 9 1.75 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 113 Table 3. 3 (cont’d) GO:0031348_negative_regulati on_of_defense_response GO:0031408_oxylipin_biosynt hetic_process GO:0031969_chloroplast_mem brane GO:0003700_sequence- specific_DNA_binding_transcri ption_factor_activity GO:0045449_regulation_of_tra nscription 0.30 3 0.30 3 0.30 3 0.30 3 0.30 3 GO:0005667_transcription_fact or_complex 0.30 3 0.70 5 0.70 5 0.42 4 0 0 0 GO:0001053_plastid_sigma_fa ctor_activity GO:0015293_symporter_activit y GO:0007178_transmembrane_r eceptor_protein_serine/threonin e_kinase_signaling_pathway GO:0009825_multidimensional _cell_growth GO:0006598_polyamine_catab olic_process GO:0042398_cellular_modified _amino_acid_biosynthetic_proc ess GO:0006354_DNA- templated_transcription GO:0006200_obsolete_ATP_ca tabolic_process GO:0080167_response_to_karri kin GO:0016762_xyloglucan:xylog lucosyl_transferase_activity GO:0006073_cellular_glucan_ metabolic_process GO:0006813_potassium_ion_tr ansport GO:0004707_MAP_kinase_acti vity GO:0006413_translational_initi ation GO:0010264_myo- inositol_hexakisphosphate_bios ynthetic_process 0 2.71 2 0 2.71 2 0 2.23 5 0 1.69 3 0 1.50 4 0 1.50 4 0 1.50 4 0 1.40 3 0 1.35 3 0 1.35 3 0 1.35 3 0 1.35 3 0 1.35 3 0 1.35 3 0 1.35 3 0.40 2 0 - 0.10 6 0.86 8 0.92 5 0.84 6 0.23 6 0.67 5 1.17 7 1.02 9 0.16 0.52 6 0.16 0.98 - 0.27 3 - 0.44 4 - 0.44 4 0.89 2 1.47 7 2.26 4 0.72 5 0.42 0 0.42 1.76 4 1.42 6 1.62 3 3.66 2 3.59 9 3.12 3 0.49 6 0.22 4 0.52 7 0.46 4 0.99 2 0.99 2 0.75 1 0 - 0.10 4 3.04 1 2.97 5 2.73 5 0.71 8 2.02 8 1.99 1 1.98 6 0.70 3 0.70 3 1.22 4 0.30 7 0.10 1 1.03 3 0.91 6 0.91 6 0.78 6 2.59 0.66 1.22 8 0.79 9 0.79 9 0 0 0.42 0 0.59 0.76 0.45 9 1.09 6 1.09 6 0.88 7 0.45 9 0 0 - 0.35 5 1.98 5 1.41 4 1.41 4 0.61 8 0.61 8 0.49 6 0.47 5 1.18 4 0.59 6 0.29 9 0.29 9 1.47 7 1.18 5 0.29 9 3.19 7 0.11 4 0.71 8 1.58 6 1.58 6 0 0.25 0 0.23 4 0 0.78 6 1.32 6 1.32 6 0.79 8 0.54 6 0.28 2 2.33 0.66 6 0.92 5 0.86 6 2.50 6 2.66 6 2.29 3 0.72 2 1.09 8 0.51 0.38 5 1.17 8 0.94 0.50 7 - 0.59 2 0.94 0.71 5 0.71 5 0.16 7 0.16 9 0 0 0 0 0 2.14 4 1.45 1 1.34 6 4.38 9 1.15 2 1.94 1 0.79 9 1.57 2 3.54 3.60 6 4.16 3 1.36 3 2.94 6 0.76 8 0.62 7 0.24 1 0.08 9 0.76 8 0.76 8 0.25 - 0.44 1 0.98 2 0.36 3 0.36 3 0.36 3 0 0 0 3.69 5 1.35 8 0.79 1 0 0.59 9 0.11 7 1.18 7 1.18 7 - 0.18 5 - 0.59 6 1.97 9 2.40 7 2.40 7 0.51 0.24 5 0.39 5 0.64 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 114 Table 3. 3 (cont’d) GO:0015299_solute:proton_ant iporter_activity GO:0006865_amino_acid_trans port GO:0000280_nuclear_division GO:0009607_response_to_bioti c_stimulus GO:0010311_lateral_root_form ation GO:0048829_root_cap_develop ment GO:0010143_cutin_biosyntheti c_process GO:0030139_endocytic_vesicle GO:0009888_tissue_developme nt GO:0016747_transferase_activi ty GO:0008289_lipid_binding GO:0004497_monooxygenase_ activity GO:0043086_negative_regulati on_of_catalytic_activity GO:0005615_extracellular_spa ce GO:0008447_L- ascorbate_oxidase_activity GO:0010015_root_morphogene sis GO:0042742_defense_response _to_bacterium GO:0006564_L- serine_biosynthetic_process GO:0004617_phosphoglycerate _dehydrogenase_activity GO:0009767_photosynthetic_el ectron_transport_chain GO:0010315_auxin_efflux GO:0009768_photosynthesis GO:0006629_lipid_metabolic_ process GO:0004871_signal_transducer _activity 0 1.35 3 0 1.35 3 0 1.35 3 0 1.35 3 0 1.35 3 0 1.35 3 0 1.35 3 0 1.35 3 0 1.35 3 0 0.94 2 0 0.77 8 0 0.70 5 0 0.70 5 0 0.70 5 0 0.67 6 0 0.67 6 0 0.67 6 0 0.67 6 0 0.67 6 0 0.67 6 0 0.67 6 0 0.67 6 0 0.54 5 0 0.42 4 0.27 0.27 0.27 0.22 4 0 0 0 0 - 0.17 3 1.41 4 1.80 7 2.20 3 1.81 9 1.48 8 2.98 1.83 8 1.74 9 1.48 8 1.48 8 1.48 8 1.48 8 1.48 8 1.42 6 1.42 6 1.18 5 0.59 6 0.29 9 0.29 9 0.29 9 0.59 6 0.59 6 0.29 9 0.59 6 0.64 8 0.39 7 0.93 1 0 0 0.29 9 0.59 6 3.19 7 0.59 6 0.59 6 0 0 0 0.45 0 0.67 5 0.23 6 0.22 6 0.22 6 0.88 7 0.22 6 0.22 6 0 0.44 1 0.16 0.09 5 0.40 8 0.40 8 - 0.18 0.65 0.88 7 1.93 5 0 0 0 0 0 0.35 2 - 0.10 6 0.25 0.48 1 0.71 8 0.69 2 0.94 0.48 1 0.25 0.25 0.69 2 0.16 7 0.45 7 0.27 8 0.90 8 0 1.17 1 0.69 2 2.06 4 0 0.54 6 0.54 6 0.28 2 0.28 2 0.54 6 0.54 6 0.28 2 0.28 2 0.28 2 0.38 4 0.11 1 0.47 8 0.47 8 0 0.54 6 1.32 6 4.37 6 0 0 0 0.25 0.25 0 0.64 9 0 0.78 6 0 0.28 2 0.56 9 - 0.11 9 0 0.52 5 0.52 5 0.27 9 0 0.94 4 -0.3 0 - 0.45 4 0.52 5 1.44 4 1.44 4 0.65 9 0.48 1 1.62 8 0.90 2 0.41 0.25 2 0.25 2 0.48 4 0.48 1 0.48 4 1.45 2 1.09 8 0 0 0.24 5 0 0.56 0.29 3 0 0.26 3 0.17 8 0.26 3 - 0.47 9 0.95 7 0.98 4 1.72 1 1.02 8 1.00 8 1.73 6 1.37 4 1.25 8 0.25 5 0.25 5 0.25 5 0.76 8 0.51 6 2.13 8 0.45 8 0 0 - 0.28 5 0 0 0 - 0.14 9 3.11 9 1.73 5 1.45 1 1.47 4 1.58 3 2.37 6 1.71 4 1.08 7 1.18 7 1.18 7 1.18 7 1.18 7 1.18 7 2.02 0.90 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 115 Table 3. 3 (cont’d) GO:0045490_pectin_catabolic_ process GO:0051258_protein_polymeri zation GO:0006782_protoporphyrinog en_IX_biosynthetic_process GO:0006563_L- serine_metabolic_process GO:0046983_protein_dimerizat ion_activity GO:0005618_cell_wall GO:0030599_pectinesterase_ac tivity GO:0008283_cell_proliferation GO:0004857_enzyme_inhibitor _activity GO:0009505_plant- type_cell_wall GO:0009654_photosystem_II_ oxygen_evolving_complex GO:0009538_photosystem_I_re action_center GO:0009664_plant- type_cell_wall_organization GO:0042545_cell_wall_modifi cation GO:0019898_extrinsic_compon ent_of_membrane GO:0042973_glucan_endo-1 GO:0004190_aspartic- type_endopeptidase_activity GO:0042549_photosystem_II_s tabilization GO:0031072_heat_shock_prote in_binding GO:0045330_aspartyl_esterase _activity GO:0042546_cell_wall_biogen esis GO:0019685_photosynthesis GO:0016630_protochlorophylli de_reductase_activity 0 0.42 3 0 0.42 3 0 0.42 3 0 0.42 3 0 0.42 3 0 0.39 4 0 0.38 9 0 0.29 4 0 0.29 4 0 0.29 4 0 0.16 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1.62 3 1.48 8 1.48 8 1.42 6 1.35 2.16 3 2.22 7 2.19 9 1.48 8 1.46 6 4.42 8 4.07 9 3.97 6 2.85 7 2.85 7 2.60 9 2.48 2 2.04 7 1.98 5 1.83 8 1.81 9 1.80 7 1.48 8 1.48 8 0.66 7 0 0 0.16 0.16 0 2.31 1 0.55 0.26 5 0.12 8 0.69 0 0 0.87 5 0.2 0 0 0.39 3 0 0 1.09 6 0 0 0 0 0.88 3 0 0.20 2 1.86 5 0 0.09 9 0.52 6 0 0 0.09 9 1.47 7 0 0.22 8 0 0.20 2 - 0.37 1 - 0.13 0 0.59 6 0.29 9 - 0.66 5 0 0 1.28 6 0.34 5 0 0 0.52 5 1.38 3 3.11 4 0.75 1 0.92 2 0.13 1 2.15 5 0.94 0.94 1.81 5 0.45 7 0.70 3 0.21 2 0.53 7 0.23 4 0.25 1.39 0.27 8 0.48 1 0.48 1 0.59 0.59 0 0 0.59 0.64 2 1.27 1.22 8 1.03 8 0.30 4 0.19 4 3.36 9 1.32 6 0.21 2 0.11 1 2.12 7 - 0.12 8 0 0.79 8 0.78 6 1.32 6 0 0.54 6 0.54 6 0.58 5 0.72 2 1.38 9 0 0.10 2 3.66 7 1.40 1 1.01 9 6.71 2 0.37 2 2.30 4 1.46 9 0.94 1.73 9 0.56 9 0.92 5 2.71 1 0.53 8 0.48 1 0.34 6 0.37 3 0.42 4 0.94 4 0.94 4 1.07 8 0.76 8 1.24 4 0.13 8 0.22 4 3.14 4 2.09 9 1.07 8 4.35 4 0.54 4 3.36 7 2.01 2 1.24 4 2.65 7 0.98 8 1.54 9 2.68 8 1.44 8 0.76 8 0.75 6 1.02 8 1.54 9 0.76 8 0.76 8 1.34 6 1.18 7 1.18 7 0.90 2 0.88 3 2.76 9 2.19 9 1.83 8 1.97 9 0.99 6 3.47 2 3.11 9 3.17 2.05 9 2.05 9 1.73 5 1.97 9 2.14 4 1.58 3 1.71 4 1.75 1.17 3 1.18 7 1.18 7 0 0 0 0 0 0.40 2 0.21 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 116 Table 3. 3 (cont’d) GO:0000272_polysaccharide_c atabolic_process GO:0042631_cellular_response _to_water_deprivation GO:0031977_thylakoid_lumen GO:0031225_anchored_compo nent_of_membrane GO: 0045548_phenylalanine_ammo nia-lyase_activity GO:0009800_cinnamic_acid_bi osynthetic_process GO:0005199_structural_constit uent_of_cell_wall GO:0009791_post- embryonic_development GO:0016760_cellulose_synthas e_UDP-forming_activity GO:0009069_serine_family_a mino_acid_metabolic_process GO:0043565_sequence- specific_DNA_binding GO:0050662_coenzyme_bindin g GO:0003735_structural_constit uent_of_ribosome GO:0042254_ribosome_biogen esis GO:0009117_nucleotide_metab olic_process GO:0005829_cytosol 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - 0.06 7 - 0.12 5 - 0.23 3 - 0.32 3 - 0.32 3 - 0.45 2 - 3.87 1.48 8 1.48 8 1.48 8 1.46 6 1.41 4 1.41 4 1.41 4 1.41 4 1.41 4 - 0.04 2 1.84 8 1.76 4 - 0.36 6 - 0.58 7 1.42 6 - 9.04 - 0.13 - 0.70 3 - 0.88 1 0 1.09 6 1.09 6 0.44 1 0.22 8 0.10 9 0.31 8 0.25 0 - 3.13 8 - 2.37 0 - 4.32 2 - 0.46 4 - 0.46 4 0.16 0 0 0 0 0 0 - 0.38 2 - 0.54 6 - 0.72 1 - 0.89 5 - 0.89 5 - 0.49 7 - 4.90 9 0.11 4 0 - 0.20 1 0.23 9 0 0 0.69 2 - 0.14 1 0 0.33 4 1.20 2 - 0.28 - 1.33 5 - 1.33 5 0 - 5.42 5 - 0.16 4 0 0.47 8 0 0 0 0.28 2 - 0.51 3 - 0.41 2 - 1.22 6 0.13 4 - 0.35 3 0.07 2 0 - 1.19 6 - 5.25 4 1.39 7 0.94 1.38 9 0.81 2 0.52 5 0.52 5 0.52 5 1.07 9 0.90 2 0.26 1.59 0.09 1 0.25 4 - 0.34 4 1.08 9 - 13.6 21 1.49 6 1.24 4 1.95 1 0.69 6 0.36 3 0.36 3 1.16 8 0.95 7 0.74 4 0.02 9 2.28 9 0.20 1 0.14 9 - 0.28 5 2.53 4 - 13.2 21 117 0 0 0 0 0 0 0 0 0 1.18 7 1.58 3 1.18 7 0.77 1.37 8 1.37 8 0.78 3 0.78 3 0.78 3 0.07 6 1.54 9 0.58 0 1.36 3 1.36 3 0 1.36 3 2.87 8 1.21 7 - 0.54 3 - 0.76 9 1.17 3 - 7.80 5 LITERATURE CITED 118 LITERATURE CITED Alzohairy, S.A., Hammerschmidt, R., and Hausbeck, M.K. 2017. 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A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. The Plant Cell, 20:2763-2782. Zhong, R., Richardson, E. A., & Ye, Z. H. (2007). The MYB46 transcription factor is a direct target of SND1 and regulates secondary wall biosynthesis in Arabidopsis. The Plant Cell, 19:2776- 2792. Zhou, J., Lee, C., Zhong, R., & Ye, Z. H. (2009). MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis. The Plant Cell, 21:248-266. 124 FUTURE RESEARCH In our research, we studied the onset and mechanism of age-related resistance (ARR) to Phytophthora capsici in fruits of C. moschata commercial cultivars. Two cultivars with early onset of ARR were identified and can be used by growers to limit fruit rot. Incorporating cultivars with ARR may reduce the number of fungicide applications required. Additional screening of squash and pumpkins commercial cultivars for those with early onset of ARR to P. capsici is needed. Squash and pumpkins germplasm with resistance to P. capsici are needed for breeding programs to develop varieties with fruit rot resistance. The mechanism of ARR in winter squash to P. capsici was investigated. Our results suggest that ARR in winter squash to P. capsici is conferred by a physical barrier that is formed by lignin deposition in the fruit exocarp at the cuticle and epidermal walls. Observations from the scanning electron microscope study of fruit exocarp structural defense indicated that at 24 hpi, P. capsici attempted to penetrate the resistant fruits through stomatal openings. Cell wall dissociation 24 hpi or at 48hpi suggested that a structural barrier is present at the stomatal opening. Further examination of that structural barrier should be conducted. Lipid stain (ex. sudan IV dye; stains lipids yellow) can be used to examine the suberization at the stomata by comparing young susceptible and mature resistant fruit stages inoculated with P. capsici or not inoculated. Therefore, we can detect if suberization occurs prior to P. capsici attack (preformed) or induced suberization occurs post inoculation. Lignin deposition at the fruit exocarp was detected and may confer resistance against P. capsici. A disease resistance screening method based on lignin content in the fruit exocarp could be developed. Phloroglucinol stains lignin in red, therefore lignin content can be determined in 125 proportion to the intensity of the color in the fruit peel. During fruit development, lignin concentration at which resistance occurs can be determined then used in further high-throughput phenotypic screening methods for resistance. The quantitative expression of lignin biosynthetic genes including CAD, CCR, CCoAOMT and peroxidases in the fruit exocarp could be determined using qPCR and indicate lignin biosynthesis and deposition at the fruit cell wall. If the quantitative expression of these genes can be determined in the resistant fruit stages, then these genes can be used in marker-assisted selection in breeding programs to develop squash or pumpkins varieties with resistance to P. capsici. 126