Identification And Functional Study Of Pathogenicity Genes Of Kiwifruit Canker Pathoge

Bacterial canker of kiwifruit,caused by Pseudomonas syringae pv.actinidiae（Psa）,has a devastating effect on the kiwifruit industry.Novel methods of disease control require better understanding of the bacterial pathogenic mechanism.Although some potential pathogenic factors have been identified in strains with differences in virulence,the key pathogenic factors of Psa have not yet been identified.Previous reports have identified several biovar populations of Psa with differences in genome sequence,pathogenic gene composition,and geographical distribution.However,all Psa isolates in China were classified as Psa biovar 3（Psa3）,which is made up of several genetically identical subgroups but with extensive variations in virulence among individual strains.Therefore,based on the information of population structure and virulence differentiation of Psa3 isolates from Guizhou Province,we have tried to identify the genetic variation of pathogenic genes,which are responsible for the loss and reduce of virulence,and analyze their roles in natural variation of bacterial pathogenicity.The detailed results shown below:1.Previously,377 Psa isolates were collected,and 84 of them were shown to be non-pathogenic or very low-virulent using in vitro branch inoculation tests.Here,we traced back their origins and discovered that the non-pathogenic isolates were frequently isolated.Then the genetic causes of the loss or reduction of bacterial virulence was elucidated by studying30 non-pathogenic strains and one low-virulent strain.1)The comparative genomics and PCR detection revealed that the transposon ISPsy36 inserted into hrp R gene in 22 non-pathogenic strains,leading to a functional deficiency in the type Ⅲ secretion system（T3SS）,as previously reported.2)The causes of virulence attenuation in other non-pathogenic and low-virulent isolates remain unknown.To identify the genetic causes underlying loss-of-virulence in Psa3,comparative genomics and PCR detection were performed firstly.The results showed that a novel transposon insertion within hrp S gene by ISPsy36 was detected in non-pathogenic strains G230 and G277,and expressing the hrp S gene in G230 restored its T3SS function,reestablishing its pathogenicity in host kiwifruit plants and hypersensitive response（HR）in non-host Nicotiana benthamiana plants.The results indicated that loss-of-virulence in these two strains is due to the insertion of ISPsy36 into the hrp S gene.2)A nonpathogenic isolate G166 was found to be defective in hrp L transcription and the downstream type Ⅲ secretion system（T3SS）-dependent phenotypes.Comparative genomics and complementary expression assay revealed that a single-base“G”insertion in the hrp L promoter blocks gene transcription by reducing promoter activity.The electrophoretic mobility shift assay showed that the genetic variation impairsσ⁵⁴/promoter binding during gene transcription under hrp-inducing conditions,resulting in lower expression of hrp L.A PCR-restriction fragment length polymorphism assay was performed to trace the evolutionary history of this mutation,and identified other three non-pathogenic strains,G321,G222,and G293,which contained the same genetic variation and belonged to different Psa3 subgroups,suggesting the independent onset of genetic variations in natural Psa3 populations.Besides,we found that loss-of-virulence of stain G411 strain was caused by frameshift mutation in the hrp L gene.4)The T3SS function of non-pathogenic strain G126 is deficient.The promoter activity of the hrp R/S gene was somewhat reduced in G126,whereas the transcription of hrp L gene was blocked.The transcriptome and proteome sequencing revealed that transcription level of hrp R/S and protein levels of Hrp R and Hrp S were somewhat reduced,while expression of the hrp L and downstream T3SS genes was dramatically reduced in G126.Comparative genomics revealed that Hrp R in G126 may premature or N-terminal truncated due to a single SNP in the codon encoding the 10th amino acid（R10*）.Expressing hrp R_G1 in G126 under the control of its own promoter,restored its ability to induce HR in N.benthamiana leaves,demonstrating that the SNP within the hrp R gene resulting in N-terminal truncated Hrp R protein is responsible for the inactivation of hrp L and T3SS genes,leading to a loss of virulence.5)The virulence of strain G282 was significantly diminished but not eliminated.Using comparative genomics and transcriptomics,we found that G282 lacked the type VI secretion system（T6SS）gene cluster and had drastically reduced expression of T3SS genes.It has been found that knockout of T6SS genes resulted in reduced T3SS expression and virulence in Psa3.Thus,we hypothesized that the reduced pathogenicity of G282 is due to the natural deletion of T6SS gene cluster.6)The frequently formation of non-pathogenic variants during the course of host infection by Psa3 suggest that non-pathogenic variants may have a growth advantage in the mixed populations.The non-pathogenic strains G4,G230,and G166 were found to outgrow the pathogenic strain G1 in the oligotrophic HDM medium;and the G166 outperformed virulent Psa3 bacteria for both epiphytic and apoplast colonization of kiwifruit leaves in mixed inoculations.Based on these findings,it appears that non-pathogenic variants of Psa3 originate from mutations in the T3SS regulatory genes hrp R/S and hrp L,suggesting that these mutations have played a significant role in the virulence evolution of Psa3.2.Transcriptome data showed that the expression levels of avrPto5 in the three strains（G126,G282,and G1）were highly consistent with the corresponding hrp L expression levels.And the presence of AvrPto5 in each of the five Psa biovars suggests it plays an important role in the host-pathogen interaction.Therefore,we examined the function of type Ⅲ effector AvrPto5 in bacterial pathogenicity.1)We successfully produced a non-selectable in-frame knockout mutant G1avrPto5 and tested its pathogenicity,discovering that avrPto5’s pathogenicity was greatly enhanced after knockout.Mating method screening was used to identify 12 potential target proteins after the Y2H AvrPto5 bait vector was created and found to be free of toxicity and self-activation.By using GST-pull-down,we were able to confirm in vitro that AvrPto5 interacts directly with two of the target proteins.4)Research into the target protein KWSAP5 revealed that it is a member of the zinc finger family of proteins and may have a role in plant immunity.AvrPto5 has been shown to have an effect on bacterial pathogenicity,but more research is needed to determine the molecular foundation of this interaction and the method by which it works.