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Comparative Analysis of Temporal Transcriptome Reveals The bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.323402; this version posted October 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 1 Comparative analysis of temporal transcriptome reveals the 2 relationship between pectin degradation and pathogenicity of 3 defoliating Verticillium dahliae to Upland cotton (Gossypium 4 hirsutum) 5 6 Fan Zhang1,2,#a, Jiayi Zhang1; Wanqing Chen3; Xinran Liu3; Cheng Li1; Yuefen Cao1; Tianlun 7 Zhao1; Donglin Lu1; Yixuan Hui1; Yi Zhang1; Jinhong Chen1; Jingze Zhang3; Alan E. 8 Pepper2*,#a; John Z. Yu4*; Shuijin Zhu1* 9 10 1 Institute of Crop Science, College of Agriculture & Biotechnology, Zhejiang University, 11 Hangzhou, Zhejiang, China 12 2 Department of Biology, College of Science, Texas A&M University, College Station, Texas, 13 United States of America 14 3 Institute of Biotechnology, College of Agriculture & Biotechnology, Zhejiang University, 15 Hangzhou, Zhejiang, China 16 4 United States Department of Agriculture-Agricultural Research Service (USDA-ARS), 17 Southern Plains Agricultural Research Center, College Station, Texas, United States of 18 America 19 20 #a Current Address: Department of Biology, College of Science, Texas A&M University, 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.323402; this version posted October 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 21 College Station, Texas, United States of America 22 23 * Corresponding author: 24 Email: [email protected] (A.E.P.), [email protected] (J.Z.Y.), and [email protected] 25 (S.J.Z). 26 27 Abstract 28 Verticillium wilt (VW), caused by Verticillium dahliae Kleb., is a major plant disease that 29 causes heavy annual losses around the world, especially in Upland cotton (Gossypium 30 hirsutum). The disease-causing pathogen can be classified into defoliating (D) and 31 non-defoliating (ND) pathotypes based on the induced symptoms. At present, little is known 32 about the complex mechanisms of fungal pathogenicity and cotton resistance to it. 33 Comparative analysis of temporal transcriptome was performed on two V. dahliae strains, 34 Vd_086 (D) and Vd_BP2 (ND), at key development stages (hyphal growth, microsclerotia 35 production, and spore germination) to reveal the functional process on plant defoliation and 36 death. Differentially expressed gene (DEG) analysis revealed a strong correlation between 37 cell wall protein kinase activities and the early pathogenicity of defoliating Vd_086. With 38 weighted gene co-expression network analysis (WGCNA), six specific gene modules were 39 correlated with the biological traits of the fungal samples. Functional enrichment with Gene 40 Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways together 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.323402; this version posted October 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 41 with DEG analysis revealed six pectin degrading enzymes including Polygalacturonase 42 gene 1 (PG1), Pectate lyase gene (PEL) and Pectinesterase gene 1 (PME1) expressed in 43 the early development of Vd_086 that may be related to the robust pathogenicity of this 44 strain during the early invasion. The expression of four of these genes was verified by 45 real-time quantitative reverse transcription PCR (qRT-PCR). In addition, we identified 46 Mitogen-Activated Protein Kinase (MAPK) signaling “hub” genes that may regulate these 47 pectinases. In a word, enhanced expression of pectin degradation enzymes is associated 48 with the stronger pathogenicity of Vd_086 than Vd_BP2, especially at early infection stages. 49 The disease-causing capability is likely regulated by MAPK signaling genes. This study 50 provides new insight into molecular mechanisms of the plant-pathogen interaction on the 51 VW disease, facilitating more effective control measures against this pathogen, including 52 molecular breeding for the VW-resistant cotton cultivars. 53 Key words: Fungal disease; Verticillium wilt (VW); Upland cotton (Gossypium hirsutum); 54 Transcriptome sequencing; Differentially expressed genes (DEGs); Weighted gene 55 co-expression network analysis (WGCNA), and Plant cell wall degradation 56 57 Author summary 58 Verticillium wilt (VW), caused by fungal pathogen Verticillium dahliae (Vd), is arguably the 59 most devastating disease in cotton production for decades. Molecular biologists and plant 60 breeders have been working hard to identify host plant resistant genes for many years but 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.323402; this version posted October 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 61 have met with little success due to the large complex genome of cotton. The V. dahliae 62 strains are grouped in two pathotypes, of which defoliating (D) strains cause total leaf loss of 63 infected cotton plants and non-defoliating (ND) strains do not. Comparative transcriptome 64 analysis of D strain Vd_086 and ND strain Vd_BP2 identified the candidate genes and 65 molecular mechanisms related to the Vd pathogenicity. Besides the difference in 66 pathogenicity, these strains are distinguishable by the rate of hyphal elongation, 67 microsclerotia production, and spore germination. With these phenotypes, transcriptome 68 sequencing of both strains was performed at the three growth phases. By the combination of 69 comparative transcriptomic differentially expressed gene (DEG) analysis and weighted gene 70 correlation network analysis (WGCNA), cell wall-associated pectinase genes were found to 71 be active at hyphal elongation stage of the V. dahliae pathogen and ribosome-related 72 processes were activated for microsclerotia production. Gene modification processes were 73 activated with many protein kinases at spore germination stage that for the next infection 74 cycle. Furthermore, four pectinases in the pentose and glucuronate interconversion (PGI) 75 pathway were identified and verified as highly expressed in the D strain with strong 76 pathogenicity to Upland cotton (Gossypium hirsutum). Our results provided evidence in 77 support of the hypothesis that stronger early pathogenicity of the D strain is resulted from 78 greater plant cell wall pectin degradability. Transcription factors (TFs) and “hub” module 79 genes were identified in searching of protein interaction for possible regulators of the 80 recognized pectinases. TFs involved in mitogen-activated protein kinase (MAPK) signaling 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.323402; this version posted October 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 81 pathway were shown to regulate not only hyphal processes but also the entire growth period 82 of V. dahliae. This is the first study known to use module extraction techniques of WGCNA to 83 identify differentially co-expressed genes between two fungal pathotypes of V. dahliae 84 strains. The study provides new insights into molecular mechanisms of the plant-pathogen 85 interaction and may lead to molecular breeding for resistant cotton cultivars to effectively 86 control this devastating disease. 87 88 Introduction 89 Verticillium wilt (VW) diseases in crop plants are spreading worldwide, especially where 90 susceptible crops are grown. They affect agricultural economies of most countries to various 91 extents [1]. Major crops affected by VW diseases include cotton, potato, tomato, strawberry, 92 eggplant, olive, sunflower, tobacco, and many herbaceous and woody perennials [2, 3]. 93 Among all these plant species, cotton (Gossypium spp.), the leading natural fiber crop, 94 suffers the most severe economic losses [4]. Of four cultivated cotton species, the disease 95 primarily affects Upland cotton (Gossypium hirsutum L) which produces approximately 95% 96 world's natural fiber. 97 98 The causal agent is Verticillium dahliae Kleb, a soil-borne fungus capable of infecting plant 99 roots and colonizing the vascular tissues throughout growth season, which was first reported 100 more than a century ago [5]. In general, V. dahliae is classified into two pathotypes: 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.02.323402; this version posted October 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 101 defoliating (D) and non-defoliating (ND), based on induced symptoms [6]. The D pathotype is 102 highly virulent and usually causes a severe wilting syndrome including chlorosis, fall of green 103 leaves (defoliation), vascular discoloration, and death of the plant. The ND pathotype does 104 not cause such severe phenotype over the same infection period (Fig. 1A, B). Infected 105 plants exhibit symptoms of decreasing in photosynthesis and increasing in respiration, 106 resulting in a significant reduction of the plant biomass and heavy loss of yield [7]. In addition 107 to the different pathogenetic capabilities, the two strains show different growth phenotypes 108 outside of the host, when cultured on Potato Dextrose Agar (PDA) plates (Fig.
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