Function Analysis Of Pectin Methylesterase During The Interaction Between Banana And Fusarium Oxysporum F. Sp. Cubense Race1 And Race4

Musa spp.is one of the major fruits in the world,and is also regarded as the forth-largest crop in the developing countries.However,banana production is affected by different factors,in which banana wilt caused by Fusarium oxysporum f.sp.cubense?Foc?is one of the important treats.Initially,Foc race1?Foc1?almost devastated the production of?Gros Michel‘.With the discovery and popularization of the resistant?Cavendish‘?AAA?subgroup of cultivars,banana industry can revive again.However,in the middle and late of the 20^th century,the appearance of race 4 of the pathogen resulted in the great changelles of banana industry once again.The study of the pathogenic differences between Foc1 and Foc4 and the molecular mechanism of different pathogenicity may provide a new theoretical basis for the resisitance of mechanism of banana.To understand the differential expression genes of roots infected by Foc1 and Foc4,based on the RNA-seq analysis,we monitored the gene expression profile changes in Brazilian banana roots at the time points 6 h,12 h,24 h and 48 h post Foc1 and Foc4infection,respectively,and further compared the the difference of gene expression in roots infected by Foc1 and Foc4.According to these differential expression genes,we screened the genes of pectin methylesterases?PME?,and further verified the change trend of PME using the method of immunofluorescence analysis.Then we obtained gene sequences of 2PME in Foc.Major research results were as follow:1.Analysis and screen the differential expression genes?DEGs?of roots infected by Foc1 and Foc4 using digital gene epression profiles.The Brazilian banana roots were harvested at 0 h,6 h,12 h,24 h and 48 h after infection and performed the analysis of RNA-seq.The roots of 0 h were used as control.The analysis results revealed 2056,4835,6319 and 4703 DEGs were up-regulated and then 3096,3117,3692 and 3761 DEGs were down-regulated in Brazilian banana roots infected by Foc1,respectively.While in Brazilian banana roots infected by Foc4,the number of up-regulated DEGs was respectively 2473,2649,3574 and 5014.And 3593,2333,9613 and 3215 DEGs were down-regulated in Brazilian banana roots infected by Foc4.These results suggested that earlier perception of pathogen invasion and a stronger induced defense response occurs in Brazilian banana roots due to Foc1 treatment than Foc4.Defense genes associated with cell wall synthesis related enzymes,pattern recognition receptor,transcription factor WRKY22,WRKY33,TGA were expressed stronger in Brazilian banana roots infected by Foc1 than Foc4,explaining that these genes involved in the defense responses of banana against Foc.These genes CNGCs and Rboh,regulating hypersensitive reaction?HR?,were up-regulated in roots infected by Foc4,but no significant change in roots infected by Foc1,which may be important to Foc4with a strong virulent and saprophytic nutrition.In addition,these genes expression of cell wall degrading enzymes PME and PG also were higher in Brazilian banana roots infected by Foc4 than Foc1,indicating that Foc4 can degraded cell wall of roots faster than Foc1 to facilitate the invasion and colonization of pathogen.Our research preliminarily explained the anti-Foc mechanism of Brazilian banana and provided a basis for futher understanding the molecular mechanism of the pathogenic differences between Foc1 and Foc4.2.PME plays a important role in Brazilian banana defense responses against Foc.We measured the content of PME and the degree of pectin methylesterification?DM?in the roots of Brazilian banana infected by Foc1 and Foc4 respectively,using chemical method and immunofluorescence labeling method.The results of chemical method showed that PME activities were lower in the roots infected by Foc1 than those in roots inoculated with Foc4,and the DM of the roots infected with Foc1 was higher than that with Foc4.Furthermore,the results of immunofluorescence labeling method indicated that the banana infected with Foc1 showed higher abundance of highly DMs of HGs,relatively higher pectin DMs and higher content of Ca²⁺-bridged pectin than that with Foc4.These results might be an element of the weak pathogenicity of Foc1.3.Oligogalacturonides?OGs?can induce the strong defense responses of banana.We measured the expression levels of six defense related genes,the callose contents,the flavonoids contents and the H₂O₂ contents in the roots of Brazilian banana treated with OGs.We found that OGs,Foc1 and Foc4 can induce the up-regulated expression of the defense-related genes and can increase the callose and flavonoid contents,and the range of the increase induced by Foc1 was higher than that induced by Foc4.These results suggested that Foc1 can induce stronger immunse responses in the roots of Brazilian banana than Foc4.We also measured that Foc4 can induce the stronger H₂O₂ outburst than Foc1,but no significant change of H₂O₂ was induced by OGs.The result indicated that the higher H₂O₂content induced by Foc4,as necrotrophic fungus,might be an important means of its strong pathogenicity.Banana roots pre-treated with OGs can be induced significantly higher defense responses by Foc4 than no pre-treatment with OGs,inducating that OGs can induce defense responses of banana.These results aslo suggested that the content of OGs might be may be involved in the regulation of the degree of immune responses on infected banana plants with Foc1 and Foc4.4.Foc can release PME to degrade the plant cell wall.We cloned two PME genes of Foc1 and Foc4,respectively,and performed a series of information comparison.We found that there were 16 different nucleotide sites,1 different amino acid site,99.75%amino acid sequence similarity between Foc1-g10008052 and Foc4-g10004388.Between Foc1-g10006977 and Foc4-g10009887,there were 31 nucleotide differences,resulting in 9amino acid sequence differences and 97.31%amino acid sequence similarity.Then,we detected respectively the genes expression level of PME in Foc1 and Foc4 treated with glucose,cell wall extract,methyl-free pectin and methyl esterified pectin?55%-65%?.In all cases,the degree of up-regulated of PME was significantly higher in Foc4 than that in Foc1,suggesting Foc4 can produce more PME to degrade cell wall than Foc1.