Abstract：Apple tree canker caused by Valsa mali seriously restricts the development of apple industry in China. It is of great theoretical significance for the effective prevention and control of apple tree canker to comprehensively analyze the pathogenic molecular mechanism of V. mali. The pH-responsive transcription factor PacC play an important role in the infection and pathogenic process of V. mali. In order to reveal PacC transcriptional regulation mechanism, the gene expression profiles of V. mali wild-type 03-8 and the PacC deletion mutant ΔVmPacC on branches were analyzed by RNA-seq assays. The results showed that there were 238 and 449 differentially expressed genes (DEGs) during pre-infection and post-infection of V. mali. GO enrichment analysis showed that biological processes of DEGs were mainly involved in carbohydrate metabolic processes, oxidation-reduction process, transmembrane transport process and polysaccharide metabolic processes. KEGG functional enrichment analysis showed that the pathways of DEGs were mainly involved in starch and sucrose metabolism, cyanoamino acid metabolism, pentose and glucuronate interconversions, these DEGs involved in three pathways were mainly related to plant cell wall polysaccharide (PCWP) degradation. The amino acid sequences of DEGs involved PCWP degradation were submitted to Pathogen Host Interactions (PHI)-base blast, that obtained 3 potential pathogenic related genes including putative pectate lyase A gene, pectin lyase B gene, hypothetical protein gene VM1G_08430. In this study, six DEGs were selected for qRT-PCR verification, and the results showed that the mRNA expression patterns of the 6 genes were consistent with the results of RNA-seq assays. The comprehensive analysis showed that transcription factor PacC functions in the infection and pathogenic process by regulating the expression of PCWP degradation genes. The study revealed PacC transcriptional regulation mechanism in the infection and pathogenic process of V. mali, and it would lay a theoretical foundation a theoretical basis for explaining the pathogenic mechanism of V. mali.
[1] 曹克强, 国立耘, 李保华, 等. 2009. 中国苹果树腐烂病发生和防治情况调查[J].植物保护, 35(2): 114-117. (Cao K Q, Guo L Y, Li B H, et al.2009. Investigations on the occurrence and control of apple canker in China[J]. Plant Protection, 35(2): 114-117.) [2] 王建华. 2012. 苹果树腐烂病菌致病物质的初步研究[D]. 硕士学位论文, 西北农林科技大学, 导师: 王慧, 黄丽丽, pp.18-21. (Wang J H.2012. Studies on the pathogenic substances produced by Valsa mali[D]. Thesis for M.S., North West Agriculture and Forestry University, Supervisor: Wang H, Huang L L, pp. 18-21.) [3] Alkan N, Espeso E A, Prusky D.2013a. Virulence regulation of phytopathogenic fungi by pH[J]. Antioxidants and Redox Signaling, 19(9): 1012-1025. [4] Alkan N, Meng X, Friedlander G, et al.2013b. Global aspects of pacC regulation of pathogenicity genes in Colletotrichum gloeosporioides as revealed by transcriptome analysis[J]. Molecular Plant-Microbe Interactions, 26(11): 1345-1358. [5] Barda O, Maor U, Sadhasivam S, et al.2020. The pH-responsive transcription factor PacC governs pathogenicity and ochratoxin A biosynthesis in Aspergillus carbonarius[J]. Frontiers in Microbiology, 11: 210. [6] Caracuel Z, Roncero M I, Espeso E A, et al.2003. The pH signalling transcription factor PacC controls virulence in the plant pathogen Fusarium oxysporum[J]. Molecular Microbiology, 48(3): 765-779. [7] Chen Y, Li B, Xu X, et al.2018. The pH-responsive PacC transcription factor plays pivotal roles in virulence and patulin biosynthesis in Penicillium expansum[J]. Environmental Microbiology, 20(11): 4063-4078. [8] Chou C, Yu F, Yu P, et al.2015. Expression of five endopolygalacturonase genes and demonstration that MfPG1 overexpression diminishes virulence in the brown rot pathogen Monilinia fructicola[J]. PLOS ONE, 10(6): e0132012. [9] Eshel D, Miyara I, Ailing T, et al.2002. pH regulates endoglucanase expression and virulence of Alternaria alternata in persimmon fruit[J]. Molecular Plant-Microbe Interactions, 15(8): 774-779. [10] Hu Y, Lian L, Xia J, et al.2020. Influence of PacC on the environmental stress adaptability and cell wall components of Ganoderma lucidum[J]. Microbiological Research, 230: 126348. [11] Isshiki A, Akimitsu K, Yamamoto M, et al.2001. Endopolygalacturonase is essential for citrus black rot caused by Alternaria citri but not brown spot caused by Alternaria alternata[J]. Molecular Plant-Microbe Interactions, 14(6): 749-757. [12] Kanda S, Aimi T, Kano S, et al.2008. Ambient pH signaling regulates expression of the serine protease gene (spr1) in pine wilt nematode-trapping fungus, Monacrosporium megalosporum[J]. Microbiological Research, 163(1): 63-72. [13] Kanehisa M, Araki M, Goto S, et al.2008. KEGG for linking genomes to life and the environment[J]. Nucleic Acids Research, 36(Database issue): D480-484. [14] Kubicek C P, Starr T L, Glass N L.2014. Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi[J]. Annual Review of Phytopathology, 52: 427-451. [15] Kunitake E, Hagiwara D, Miyamoto K, et al.2016. Regulation of genes encoding cellulolytic enzymes by Pal-PacC signaling in Aspergillus nidulans[J]. Applied Microbiology and Biotechnology, 100(8): 3621-3635. [16] Landraud P, Chuzeville S, Billon-Grande G, et al.2013. Adaptation to pH and role of PacC in the rice blast fungus Magnaporthe oryzae[J]. PLOS ONE, 8(7): e69236. [17] Lopez-Perez M, Ballester A R, Gonzalez-Candelas L.2015. Identification and functional analysis of Penicillium digitatum genes putatively involved in virulence towards citrus fruit[J]. Molecular Plant Pathology, 16(3): 262-275. [18] Luo Z, Ren H, Mousa J J, et al.2017. The PacC transcription factor regulates secondary metabolite production and stress response, but has only minor effects on virulence in the insect pathogenic fungus Beauveria bassiana[J]. Environmental Microbiology, 19(2): 788-802. [19] Lysøe E, Pasquali M, Breakspear A, et al.2011. The transcription factor FgStuAp influences spore development, pathogenicity, and secondary metabolism in Fusarium graminearum[J]. Molecular Plant-Microbe Interactions, 24(1): 54-67. [20] Manteau S, Abouna S, Lambert B, et al.2003. Differential regulation by ambient pH of putative virulence factor secretion by the phytopathogenic fungus Botrytis cinerea[J]. FEMS Microbiology Ecology, 43(3): 359-366. [21] Merhej J, Richard-Forget F, Barreau C.2011. The pH regulatory factor Pac1 regulates Tri gene expression and trichothecene production in Fusarium graminearum[J]. Fungal Genetics and Biology, 48(3): 275-284. [22] Prusky D, McEvoy J L, Saftner R, et al.2004. Relationship Between Host Acidification and Virulence of Penicillium spp. on apple and Citrus fruit[J]. Phytopathology, 94(1): 44-51. [23] Rascle C, Dieryckx C, Dupuy J W, et al.2018. The pH regulator PacC: A host-dependent virulence factor in Botrytis cinerea[J]. Environmental Microbiology Reports, 10(5): 555-568. [24] Shieh M, Brown R L, Whitehead M P, et al.1997. Molecular genetic evidence for the involvement of a specific polygalacturonase, P2c, in the invasion and spread of Aspergillus flavus in cotton bolls[J]. Applied and Environmental Microbiology, 63(9): 3548-3552. [25] Urban M, Cuzick A, Rutherford K, et al.2017. PHI-base: A new interface and further additions for the multi-species pathogen-host interactions database[J]. Nucleic Acids Research, 45(D1): D604-D610. [26] Wang Y, Liu F, Wang L, et al.2018. pH-signaling transcription factor aopacc regulates ochratoxin a biosynthesis in Aspergillus ochraceus[J]. Journal of Agricultural and Food Chemistry, 66(17): 4394-4401. [27] Wu Y, Yin Z, Xu L, et al.2018. VmPacC Is required for acidification and virulence in Valsa mali[J]. Frontiers in Microbiology, 9: 1981. [28] Yakoby N, Beno-Moualem D, Keen N T, et al.2001. Colletotrichum gloeosporioides pelB is an important virulence factor in avocado fruit-fungus interaction[J]. Molecular Plant-Microbe Interactions, 14(8): 988-995. [29] Yin Z, Ke X, Huang D, et al.2013. Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR[J]. World J Microbiol Biotechnol, 29(9): 1563-1571. [30] Young M D, Wakefield M J, Smyth G K, et al.2010. Gene ontology analysis for RNA-seq: Accounting for selection bias[J]. Genome Biology, 11(2): R14. [31] Zhang T, Sun X, Xu Q, et al.2013. The pH signaling transcription factor PacC is required for full virulence in Penicillium digitatum[J]. Applied Microbiology and Biotechnology, 97(20): 9087-9098.
