| Banana(Musa spp.)is one of the most important fruit crops in the world.Like other crops,the cultivation of banana is also limited by many factors.Among them,banana fusarium wilt caused by Fusarium oxysporum f.sp.cubense(Foc)is the most principle limiting factor of banana production.Initially,the banana fusarium wilt caused by Foc1 almost destruction of the ‘Gros Michel’.The banana industry was revived with the continuous discovery and promotion of resistant varieties.However,in the middle of the last century,the emergence and spread of banana fusarium wilt caused by the invasive race of Foc4 once again broke the banana industry’s situation and faced greater challenges.Previous studies have shown that both Foc1 and Foc4 can infect the Cavendish banana variety‘Brazilian’,but only Foc4 can cause disease,while Foc1 does not cause disease.However,it is still unclear why this difference has occurred.In order to clarify the resistance difference between Foc1 and Foc4 in ‘Brazilian’,we used both GFP-tagged Foc1 and Foc4 to infect ‘Brazilian’,and then used confocal LSM to observe the infection difference in ‘Brazilian’ different parts after two pathogens inoculation.We then selected the ‘Brazilian’ root sample at 48 h after inoculation for TMT-based quantitative proteomics and transcriptome sequencing analysis.Correlation analysis of differential gene expressions from proteomics and transcriptome,from which we screened a large number of potential plant resistance related genes,and we selected four genes for preliminary functional studies.The main findings are as follows:1.Through histological observation and analysis after pathogen infection,we conclude that both Foc1 and Foc4 can infect ‘Brazilain’,but the infection rate,hyphae enrichment,expansion range and the amount of fusaric acid(FA)produced in infecting host has a significantly differed after Foc1 and Foc4 Infection.Foc1 can expand from the root to the rhizome,and the biomass of the pathogen in the rhizome was significantly lower than that of the pathogen in the rhizome after Foc4 infection.While Foc4 can expand from its roots to rhizomes,to pseudostems,and finally to leaf sheaths and leaf tissue.2.Through TMT-based quantitative proteomic analysis,a total of 7325 proteins were confidently identified,and the differentially expressed protein was screened with p≤0.05,fold change ≥1.2 as the threshold.After Foc1 infection,there were 689 protein differential expressions compared with the control.Among them,463 were up-regulated and 226 were down-regulated,after Foc4 infection,744 proteins were differentially expressed compared with the control,of which 450 were up-regulated and 294 were down-regulated,interestingly,compared with Foc4 infection,a total of 1222 proteins were differentially expressed after Foc1 infection,of which 659 were up-regulated and 563 were down-regulated.RT-q PCR and PRM validation were consistent with proteomic quantification results.Bioinformatics analysis showed that redox balance,pathogenesis-related proteins,plant cell wall modifications,plant hormones and signal transduction,pattern recognition receptors,plant secondary metabolites,post-translational modifications,and programmed cell death related proteins were involved in Foc infection response.However,the expression patterns of these proteins after Foc1 and Foc4 infection are different,which may be related to the different defense mechanisms of ‘Brazilian’ against to both Foc races.3.By comparing transcriptomics analysis of the same batch of samples,we demonstrated changes on gene expression and pathways in ‘Brazilian’ during early infection with Foc using transcriptome analysis.We identified 1862 and 226 differentially expression genes(DEGs)in Foc1 infected roots and Foc4 infected roots at 48 h post-inoculation,respectively.Flavonoids and the lignin synthesis pathways were enhanced,glucosinolates,terpenoids,and proanthocyanidins accumulated and many hormonal-and receptor-kinase related genes were expressed,caused by Foc infection during its early phase.Therefore,the early interaction between ‘Brazilian’ and Foc plays an important role in the entire infection process.Furthermore,the DGEs involved in pathways related to biotic stress also differed substantially between the two Foc infection.Further analysis indicated that the genes involved in cell wall metabolism and phenylpropanoid metabolism were differentially expressed in the progression of the disease.4.Quantitative proteomics data and transcriptome sequencing data were correlated.In the three comparison groups,the number of related differential genes was 108,14 and 146,and the correlation coefficients of the two groups were 0.211,0.152 and 0.263,respectively.Bioinformatics analysis found that genes in pathways related to plant disease resistance,such as phenylpropanoid biosynthesis,flavonoid biosynthesis,glutathione metabolism,and plant and pathogen interaction,were up-regulated at both the transcriptional and protein levels in Foc1 vs.CK group,which may be related to the high resistance of ‘Brazilian’ to Foc1,and most of the genes are associated with heat shock proteins and 60 S ribosomal proteins in Foc4 vs.CK group,.five of the associated genes were not pathway annotated and were downregulated at both the transcriptional and protein levels,which may be related to ‘Brazilian’susceptible to Foc4.5.Preliminary determination of four candidate genes were associated with a difference in resistance to Foc1 and Foc4 by ‘Brazilian’.In this study,the coding sequences(CDS)of four genes were cloned,and their sequences were analyzed by bioinformatics.Plant expression vectors were constructed,and the subcellular localization of the encoded proteins was observed.A series of disease resistance experiments showed that these four candidate genes may play a pivotal role in the difference in resistance between Foc1 and Foc4 in‘Brazilian’.Our research provides a theoretical basis for the subsequent resistance breeding of bananas. |
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