Population Composition And Pathogenic Mechanism In Pseudomonas Syringae Pv.actinidiae

Bacterial canker of kiwifruit,the most destructive disease on kiwifruit,was first recorded in Japan in 1984,and has spread to all major kiwifruit cultivated areas(such as New Zealand,Italy,China,Chile,etc.)in recent years,and is threatening seriously to the global kiwifruit production.This severe disease always spread very quickly since the first break in a certain area,however,no effective solutions to the big challenge so far.The causal agent of the disease,Pseudomonas syringae pv.actinidiae(Psa),was composed of at least four genetically distinct Psa populations over the past three decades: a pandemic monomorphic population biovar 3(Psa3)and three endemic populations biovar 1,2 and 5.However,the Chinese Psa populations haven’t been clearly defined since the very early disease outbreaks in 1986.To develop novel effective strategies,there is an urgent need to know more about the pathogenic mechanisms.Several efforts have been made to unveil the pathogenicity mechanisms of Psa via different methods.However,the trigger of the full bacterial pathogenicity has not been elucidated yet.In this study,we systematically investigated the genetic diversity and pathogenicity difference of Psa strains spreading in a limited but the largest Chinese kiwifruit cultivated area(i.e.Shaanxi province),and further performed comparative studies between low-and high-virulent strains to identify the pathogenic determinants in Psa.The detailed results listed below.,1.The population composition and pathogenic diversity of Shaanxi Psa strains were uncovered.In this study,106 pure isolates were obtained from the diseased woody canes,leaves and flowers,and the roots of disease kiwifruit trees.This diseased samples were collected from eight kiwifruit cultivars and the stocks,which distributed in 90 kiwifruit orchards.These isolates were identified Psa based on the poly-phasic taxonomy methods.All of the randomly selected 12 strains fulfilled the Koch’s postulates.To unveil the population composition of Psa strains in Shaanxi,several genetic typing methods were applied.All of the20 selected Psa strains were classified into biovar 3 group,based on the results by the Rep-PCR finger-printing technique and multi-locus sequence analysis(MLSA).Whole-genome analysis of 5 strains in them showed confirmed the MLSA results.Moreover,to investigate the pathogenic diversity among Shaanxi Psa3 strains,a rapid,reliable in-door inoculation method(wound inoculation on the detached dormant woody canes)was developed.In total,30 Shaanxi Psa3 strains were wound inoculated on the detached canes of Actinidia chinensis cv.HongYang,revealing a significant pathogenic diversity.However,the pathogenicity patterns were not correlated with the sources(geographical source,host varieties and type of the diseased tissues)and the genetic populations of Psa.Remarkably,a very low virulent Psa strain M227 found in this study can be used for identification of pathogenic determinants.2.T3 SS is required for Psa pathogenicity revealed by the comparative secretome analysis and comparative genomic analysis between high-and low-virulent strains.In details,a low-virulent Psa strain M227 exhibited severely reduced pathogenicity on canes and leaves of both A.chinensis and A.deliciosa plants,and attenuated capacity to elicit the hypersensitive response(HR)in Nicotiana benthamiana leaves,compared to a high-virulent strain M228.Given to that bacterial secretome play important roles in pathogenicity,we performed a comparative secretome analysis of M227 and M228 using a mass-spectrometry-based quantitative method after growing in hrp-inducing environment,mimicking the in planta conditions.This allowed the identification of 61 proteins differentially secreted between M227 and M228(number of unique peptides ≥ 2,P<0.01),of which 40 were down-regulated and 21 were up-regulated.In the set of the most significantly different(>1.5-fold)proteins,22 and 12 proteins were down-and up-regulated,respectively.Remarkably,15 out of these 22 up-regulated proteins(meeting the 1.5-fold threshold)were found to be T3SS-associated proteins.These proteins include the most differently secreted type III harpin HopP1,translocator HrpK1,pilus HrpA1,T3 Es and their chaperones.The low pathogenicity of M227 is probably related to an impaired T3 SS.We then constructed two T3SS-deficiencent stains M228ΔhrcC and M228ΔhrcS,which caused symptom-less on kiwifruit canes and leaves and no HR in N.benthamiana leaves,indicating the important roles of T3 SS in pathogenicity.Moreover,comparative genomics between a low-virulent Psa strain M227 and two high-virulent Psa strains M228 and M401,suggested eight M227-specific variants across the 6.2 Mb genome.Via allele replacements and phenotypic analysis,we found that a naturally 3-aa deletion(65VRP67)in the small RNA chaperon Hfq in M227 resulted in compromised bacterial growth and slightly reduced pathogenicity,while a T3SS-related variation was responsible for the loss of pathogenicity and attenuated capacity to elicit HR in non-host N.benthamiana leaves.Taken together,the T3SS-related locus is important for T3 SS assembly and T3 Es secretion via a yet unknown mechanism in Psa,resulting in severely reduced pathogenicity on kiwifruit plants.3.The key T3 Es required for the pathogenicity and functional overlap among T3 Es were revealed via systematical deletions in Psa T3 E repertoire.In details,based on genomic data and secretome data,the T3 E repertoire of Psa was established,including two potential conserved effectors newly identified here.We then constructed a mutant library in deletion of the single or multiple T3 E gene(s)from two Psa strains.The mutant library is composed of 64 M228-derivative mutants and 27 M7-derivative mutants,and as many as 20 genes were deleted in a single strain.The pathogenicity of these mutants were investigated on various kiwifruit species/cultivars.Fiver novel findings were showed here: ⅰ)the core-T3 Es HopM1/AvrE1 were essentially required for Psa pathogenicity on various kiwifruit species/cultivars,and showed different evolution patterns between them;ⅱ)the variable-T3 E HopR1 was surprisingly essentially required for full virulence,but did not overlap to hopM1/avrE1 in pathogenicity;ⅲ)HopZ5 in T3 E cluster E is a member of the redundant T3 E group(cluster A,E and F)identified in this study;ⅳ)several effectors,such as AvrRpm1 and HopS2,showed inconsistent roles in pathogenicity on different kiwifruit species/cultivars;ⅴ)the deletion of each of several T3 Es,such as AvrPto5,HopAZ1,HopQ1 and HopAS1,resulted in increased pathogenicity compared to their parental strains.