| Potato(Solanum tuberosum L.)is an important crop for grain and vegetables as well as significantly industrial raw materials.Bacterial wilt,caused by Ralstonia solanacearum,is the severest bacterial disease affecting potato production.Nevertheless,the interaction mechanism between potato and R.solanacearum was little understood because of the complexity of the pathogen and the lack of resistance resources in potato species.That leads to the extremely tardiness of cultivating resistance varieties to bacterial wilt.This study firstly explored the sensitivity of potatoes to different R.solanacearum pathovars.Subsequently,the potato wild species S.albicans harboring extremely resistant and susceptible genotypes to the phylotype I R.solanacearum were screened out.By observing the HR response of the type Ⅲ secretion system mutant strain and the wild type strain,we determined that the resistance of the resistant genotype ALB28-1 to HA4-1 is triggered by effectors,i.e.ETI.Then we carried out our research from both pathogen and host aspects through the strategy of whole genome sequencing and comparative analysis of the pathogen as well as comparative transcriptome analysis of resistant and susceptible host genotypes.The details of our research are as follows:1 The pathogen aspect-whole genome sequencing and comparative analysis1.1 whole genome sequencing,annotation and virulence factor analysis of HA4-1In order to better use HA4-1 to study the interaction between R.solanacearum and potato,to expand the R.solanacearum genome database and to discover potential new important genes,we completed the whole genome sequencing and its assembly.In addition to the conventional 3.89 Mb chromosome and 1.95 Mb megaplasmid,a143 kb small plasmid,which was named p RSHA,was identified in strain HA4-1.We predicted the type Ⅲ effectors of HA4-1 by functional annotation and comparison with type Ⅲ effectors in the Ralsto T3 E database,and we found two new type Ⅲ effectors,Rip BR and Rip BS.The genome of HA4-1 was annotated,and the predicted virulence factors were analyzed via compared with GMI1000.The results showed that the homology of common virulence factors was high,while the variation of specific pathogenic factor type Ⅲ effector was large,which indicated that the different pathogenicity of the two strains was caused by effectors.1.2 Genome wide comparative analysis of closely related R.solanacearum strainsTo further narrow the scope of candidate avirulence effectors,we selected another R.solanacearum strain HZAU091,which is closely related to HA4-1(all classified to Phylotype I-14M),but has strong pathogenicity to ALB28-1,for whole-genome sequencing and comparing with HA4-1.The results showed that the difference between the two genomes is small.As for type Ⅲ effectors,only eight effectors in HA4-1 differ from in HZAU091,which are Rip BR,RS＿T3E＿Hyp6,Rip A5,Rip O1,Rip S6,Rip H1,Rip AO and Rip BS.These effectors may be the determinants of their different pathogenicity,among which the first 5 effectors differ widely.Although normal in HA4-1,they are absent or inserted by transposons in HZAU091.These different effectors are likely to be avirulence factors that trigger ETI.1.3 Preliminary analyses of candidate avirulence factorsWe selected the first five of the above-mentioned type Ⅲ effectors as candidate avirulence factors for preliminary analyses from four aspects:(1)Constructing the mutant of candidate avirulence factors via homologous recombination and testing their pathogenicity to ALB28-1;(2)Constructing the expression plasmids of candidate avirulence factors and transferring them into HZAU091,respectively,to construct functional recovery strains,then to test its pathogenicity to ALB28-1;(3)Constructing their transient expression vectors to test their lethal effects on plant cells by infiltrating the leaves of Nicotiana benthamiana;(4)Structural analyzing of candidate avirulence factors.Results showed that:(1)The Rip A5 mutant did not have any change in the pathogenicity of ALB28-1 compared with the wild-type HA4-1strain,indicating that Rip A5 is not a avirulence factor;(2)Compared with wild type HZAU091,the four derivative strains carrying the candidate avirulence factors(Rip BR,Rip O1,Rip S6 and RS＿T3E＿Hyp6)displayed reduced virulence to ALB28-1at different levels,indicating these four effectors could be recognized by their R genes of ALB28-1 and triggered resistance at different levels;(3)Rip BR and Rip O1 could quickly cause strong cell death at the infiltration sites of the N.benthamiana indicating that they alone could severely affect the physiological activities of plant cells.In conclusion,the effectors Rip BR,Rip O1,Rip S6 and RS＿T3E＿Hyp6 may be avirulence factors that mediated the ETI of ALB28-1 to HA4-1.2 The host aspect-comparative transcriptome analysisALB28-1 and ALB28-3,the resistant and susceptible genotypes of S.albicans,were inoculated with HA4-1 and then the root tissues of the two materials were taken5 days later for comparative transcriptome analysis.The results showed the differentially expressed genes were mainly enriched in several pathways including“plant-pathogen interaction”,“plant hormone signal transduction”,“phenylpropanoid biosynthesis” and so on.Differentially expression genes of the “plant-pathogen interaction” pathway are mainly involved in calcium ion signaling related genes,PTI pathway related genes and ETI pathway related genes such as the heat shock protein gene HSP83 and the NBS-LRR domain containing genes RPM1 and RPS2.Among them,calcium ion signaling-related genes were down-regulated in susceptible materials,HSP83 gene was significantly up-regulated in susceptible materials,and down-regulated in resistant materials.Most RPM1-related genes were up-regulated in resistant materials indicating that RPM1 pathway related genes were highly mobilized during the infection of S.albicans by HA4-1,and may play an important role in bacterial wilt resistance.In this study,through screening of resistant materials,the combination of potato-R.solanacearum with resistance mediated by ETI was found,which provided a good material basis for the study of resistant mechanism of potato against bacterial wilt.The strategy of comparative genomic analysis of closely related strains rapidly identified the candidate avirulence factors that triggered ETI,and the comparative transcriptome analysis of resistant and susceptible potato materials provided important clues for the rapid discovery of R genes recognizing avirulence factors.This study laid a solid foundation for the mining process of Avr-R genes during the interaction between R.solanacearum and potato and the research on the mechanism of resistance to bacterial wilt in potatoes.It is expected to provide a new idea for the breeding of resistance to bacterial wilt in potatoes. |
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