Pathogen-host Interactive Analysis Of Cotton Verticillium Wilt Based On Comparative Transcriptomic Sequencing

Cotton,as an important textile material,is a crucial economic crop in the world,and it is also an indispensable raw material for food,chemistry,medicine,light industry and national defense.Verticillium wilt is the most severe disease on cotton all around the world that affect the production every year.Due to the complicated infestation mechanism of the disease,and the difficulty of prevention,control,and eradication,it has become a hot topic for researchers in recent years.In this study,based on m RNA and small RNA transcriptome sequencing,from the perspective of pathogens,the cotton host and the information exchange between them,using bioinformatics methods to analyze the molecular characteristics of different Verticillium dahliae pathotypes,the role of small RNA in regulating the pathogenicity of pathogens and the molecular response of cotton.The potential molecular mechanism of the interaction between Verticillium dahliae and upland cotton were elucidated from a macro perspective.Main results were listed below:1.There were significant differences between defoliating and non-defoliating Verticillium dahliae strains in the abilities of hyphal elongation(H),microsclerotia production(M)and spore germination(S).Among them,the D strain showed obvious advantages in early pathogenicity.When infecting cotton at 7 dpi,the host disease index caused by the D and ND strains were 6.25 and 3.91,respectively,and at 16 dpi,the index increased to 39.84 and 26.56.In addition,compared with the ND strain,D strain had advantages in the speed of hyphae elongation and early spore germination,but was relatively slow in the production of black microsclerotia.2.Transcriptome sequencing was performed on samples from the H,M and S periods of the two pathogenic strains,an average of 6.14 Gb of filtered data(Clean Data)was obtained for each sample.After reference genome mapping,a total of 10,830 genes were annotated,of which 96.17%belonged to Verticillium dahliae.1,098,1,123 and 820 differentially expressed genes(DEGs)were identified in the three periods of H,M,and S,respectively,combined with the gene-weighted co-expression module analysis(WGCNA),the expression of pectin degrading enzymes in D strain was significantly higher than that in ND strain,which was one of the potential reasons for the early growth and invasion advantages of D strain.Transcription factor annotation and network regulation analysis results showed that the transcription factors: FACB,GA-BF and RC2 could regulate the expression of these pectin degrading enzyme genes,while multiple transcription factors in the mitogen-activated protein kinase(MAPK)signaling pathway could potentially drive gene co-expression modules related to early pathogenicity of D strain.A total of 4 genes related to the early virulence of V.dahliae D strain were identified.3.With small RNA sequencing of two strains(D and ND)of V.dahliae before and after infecting the upland cotton host,11.82 Mb of Clean Data was obtained for each sample on average.After infection,D and ND strains showed opposite trends in the different length intervals of small RNA: In the D strain,the number of short-segments increased and long segments decreased;while in the ND strain,the number of short-segment small RNAs decreased and long-segments increased.Judging by the increase in the number of small RNAs in host-specific length intervals(21 nt and 24 nt),there might be small RNA interaction in the pathogens after infecting the host.The small RNA identification showed that the small RNAs recognized in the D strain had been more fully annotated.According to small RNA expression analysis and target gene prediction,after infecting the host,4 differentially expressed small RNAs in D strain could regulate the expression of 185 target genes;while the 3 differentially expressed small RNAs in ND strain could only regulate 19 target genes.4.The susceptible and resistant upland cotton cultivars showed distinguished diseaseresistant phenotypes when faced with V.dahliae infection.One week after the inoculation of D strain,the susceptible variety EJ-1 began to show an exponential trend,while the significant onset period of the resistant variety ZZM-2 appeared about 13 days after inoculation.Transcriptome sequencing analysis was performed on the vascular bundles of the susceptible and resistant upland cotton infected by the two pathogenic strains,and the Clean Data of each sample reached 10.07 Gb.After mapping with the upland cotton standard line TM-1 reference genome,the specific comparison rate of all samples was higher than 82.46%.A total of 80,727 genes were annotated,of which 47% were annotated as upland cotton,and the other 50% were annotated as other cotton species.Through the analysis of differentially expressed genes at different infection periods(early stage: E,late stage: L)and gene set function enrichment analysis,it was found that the functional pathways that affected the resistance of upland cotton to V.dahliae were related to biological circadian rhythm regulation,galactose metabolism and glutathione Glycine metabolism.Among them,the pseudo-responder PRR,galactose metabolizing enzyme GAL,INV and glutathione transferase GST played important roles and directly affected the downstream pectin degradation capability.Four transcription factor families that responsible to plant disease resistance were identified as AP2/ERF-ERF,AUX/IAA,WRKY and Tify through transcription factor enrichment analysis.Further,with the analysis of pathway semantic similarity,it was concluded that plant hormone signaling pathways such as plant auxin signal and jasmonic acid signal were closely related to the resistance of upland cotton to Verticillium wilt.The results indicate that the difference in pathogenicity between D and ND strains is related to their early pectin-degrading enzyme activity and regulatory factors in the MAPK signaling pathway.The small RNA count,expression and their target gene regulation are also related to their pathotypes.While the resistance of upland cotton to V.dahliae is related to its biological rhythm and glutathione transferase activity.The increase of IAA and the stable content of JA may be one of the physiological basis of upland cotton resistance to Verticillium wilt.