Whole Genome Sequencing And Function Study Of T3SS Endomembrane Protein HrcV And Regulatory Protein HrpG Of Xanthomonas Arboricola Pv.pruni

Peach is one of the important fruit trees with great economic value in China.The peach bacteria shot hole caused by Xanthomonas arboricola pv.pruni（Xap）causes leaf perforation and defoliation,seriously affecting photosynthesis,resulting in weakened tree vigor and reduced fruit yield,which seriously affects the safe production of peach producing areas and brings huge economic.Xap is a gram-negative bacterium that typically transports type Ⅲeffector（T3E）into host cells through the type Ⅲ secretion system（T3SS）for pathogenic effects.However,there is no research on T3SS and T3E in Xap so far.In order to analyze the pathogenic mechanism of Xap from a molecular point of view,in this paper,the whole genome sequence of Xap 9-4 strain with strong pathogenicity was performed,and a gene knockout system for Xap was established to study the fuction of the inner membrane protein HrcV and the regulatory protein HrpG of T3SS in bacterial virulence to understand the pathogenic mechanism of Xap 9-4,which provides a theoretical basis for disease prevention and control.Whole genome sequencing was performed on Xap 9-4,and it was found that it has a linear chromosome and a circular plasmid.The genome size obtained based on the sequencing data is 5,174,371 bp,the GC value is 66.05%,and there are 4,660 genes in total;a total of 52 tRNAs,6 rRNAs and 143 sRNAs,and there are 376 tandem repeat sequences,including 159 minisatellite sequences and 108 microsatellite sequences.The synteny analysis of strain Xap 9-4 and other Xanthomonas including X.arboricola pv.corylina、X.arboricola pv.juglandis 和 X.oryzae pv.oryzae（Xoo）shows that the Xap 9-4 strain has the highest synteny with X.arboricola pv.corylina CFBP2565.Xap has not yet established a genetic operation system.In this paper,a marker-free gene knockout system based on Xap has been established for the first time,we found that the knockout vector pKMS1 suitable for X.oryzae pv.oryzae can be used in Xap.After knocking out of T3SS inner membrane protein Hrcv,the morphology and growth rate of the mutant show no difference from that of the wild type.Inoculation experiment and colonization experiment showed that the mutant completely lost its pathogenicity on peach leaves,and lost the ability to cause HR on non-host plant Nicotiana tabacum.Western blot experiments show that T3Es cannot be excreted in the ΔhrcV knockout mutant,thereby failing to inhibit host PTI,explaining the increased ROS deposition after leaf inoculation with the ΔhrcV knockout mutant.After knocking out the T3SS regulatory protein HrpG,the growth morphology and rate of the mutant in complete medium were not significantly different from those of the wild type,but its pathogenicity was significantly lower than that of the wild type.Besides,21 T3Es were found in the Xap 9-4 strain by BLAST sequence alignment qRT-PCR demonstrated that HrpG positively regulates most T3Es,thus explaining the greatly reduced pathogenicity of ΔhrpG knockout mutants.