A Systemic Screening Of Effectors In Ralstonia Solanacearum And Virulence Study Of Rip25 In Potato

Both Solanum tuberosum and Ralstonia solanacearum phylotype-IIB originated in South America and share a long-term history.As the major pathovar infecting potato,the phylotype IIB clade are widely distributed worldwide,especially at four major potato growing zones in China.Due to poor knowledge of potato bacterial wilt pathogenesis,there are no efficient means established for the disease control.The type III effectors are key factors of R.solanacearum to counteract host immunity,but the virulence mechanism of most effectors is largely unknown.Currently,more than100 effectors have been reported in the R.solanacearum species complex.Hence,a larger scale functional screening of effectors is essential for both provision of more basic information on the effector inventory and acceleration of research on host resistance.Here we built three screen systems to identify virulence effectors and study their virulence mechanism.This will provide us new insights into resistance mechanisms against potato bacterial wilt and help us to find potential approaches to combat R.solanacearum.This thesis contains following contents:1.The multiple functional screening of R.solanacearum phylotype-IIB core effectora)We inoculated potato C9701 with R.solanacearum wild type and HrcV(Type III secretion system key protein gene)mutant.The results showed Hrc V mutant neither cause any symptom in potato plants nor colonize in potato tuber.This indicates that T3 SS is indispensable for potato infection.b)To establish the multiple screening system,we first analyzed the effector repertoire of 13 sequenced phylotype IIB strains in R.solanacearum T3 E database.Thirty-three effectors,which were present more than 10 times in the 13 sequenced strains,were selected as core effectors.Previous studies have shown that 20 and four core effectors are upregulated inside plant xylem and root tissues,respectively,in comparison with their expression in rich medium.This indicates that most of these core effectors are associated with plant infection processes.c)To reveal the virulence contribution of these core effectors,three different functional screenings were performed,including virulence assays of mutants on potato,growth inhibition of yeast and transient expression in N.benthamiana.We generated a collection of effector knockout mutant.Twenty-four core effectors were successfully mutated in the reference UW551 strain.Compared with the wild-type(WT)strain,six mutants were discarded because of distinguishable differences in motility assays and growth assays in rich medium.The remaining 18 mutants were subjected to virulence assays using a susceptible potato accession,C9701.Compared with the UW551 WT control,four of these effector mutants(?rip6??rip13?? rip14 and ? rip25)showed a significant reduction in virulence.Furthermore,we used a yeast heterologous expression system.The results showed that four effectors,Rip1,Rip25,Rip55 and Rip AY,strongly inhibited yeast growth under the two conditions.However,Rip6,Rip14,Rip21 and Rip27 caused a relatively weak growth inhibition.By using transient expression in N.benthamiana,we observed that four effectors(Rip5,Rip6,Rip25 and Rip56)triggered cell death in N.benthamiana.As Rip25 is required for full virulence during potato infection,induces cell death in N.benthamiana and inhibits yeast growth,we selected Rip25 as our primary research object.2.The biochemical function study of Rip25a)To verify the virulence screening data,we generated we performed genetic complementation of the ?rip25 mutant.The results showed the rip25 mutant was delayed in virulence on both potato plant and tuber.In contrast,?rip25:rip25 enhanced recovered the virulence to WT levels,even higher in potato plant.This indicated that Rip25 played a vital role during potato infection.We also cloned three Rip25 homologous genes to verify yeast growth inhibition and cell death in N.benthamiana.The results showed homologous effectors Rip25(CFBP2957)caused yeast growth inhibition.Rip25(CFBP2957)and Rip25(PO82)both triggered cell death in N.benthamiana.This demonstrated that Rip25 could cause virulence in yeast and N.benthamiana.b)The sequence analysis showed Rip25 belonged to EspL family,a cysteine protease effector.The sequence identity between two proteins is only around30%,but the secondary structure of two protein are identical.They shared same catalytic active site in N-terminal and ankyrin domains in C-terminal.The protein tertiary structure prediction showed the structure similarity of N-terminal of Rip25 with Avrpph B,which is also a cysteine protease.The C terminal of Rip25 contained 10 ankyrin repeats.c)To study the biochemical function of Rip25,we mutated catalytic active site(C135S,D268 A,H244A,N117A)of N terminal and generated truncation of entire C terminal of Rip25.Both of them could abolish virulence function in yeast and N.benthamiana.This demonstrated that the N-terminal enzymic domain and C-terminal ankyrin domain are indispensable for the virulence.Interestingly,we observed N-terminal auto-cleavage during Rip25 expression in both yeast and N.benthamiana and this cleavage is dependent on catalytic active site.This suggested Rip25 is a cysteine protease.d)To investigate function of ankyrin repeat domains,we generated one by one truncation of repeats.The results showed all truncated proteins of ANK lost the ability to trigger cell death in N.benthamiana.It suggested that number of ANK repeats is vital for the virulence function.We also test self-cleavage site of truncated protein.The results showed three self-cleavage sites appeared during expression of truncated protein with less than 5 repeats.Comparing with wild type,the truncated Rip25 with more than 5 ankyrin repeats did not alter self-cleavage pattern.This suggested the first 5 ankyrin repeats is involved in recognition of cleavage site.Remarkably,the cleavage of all truncated protein is dependent on catalytic active site which is further confirmed that Rip25 is a cysteine protease.3.The study of cell death pathway triggered by Rip25a)To dissect cell death pathway that Rip25 triggered,we silenced immune related genes which is involved in ETI(Nb HSP9,Nb SGT1,Nb EDS1,Nb NDR1)and PTI(Nb BAK1,Nb MEKK2,Nb MAPKK,Nb WIPK)signaling pathways.The results showed that none of these genes could affect cell death triggered by Rip25,whereas silence Nb HSP90,Nb SGT1,Nb BAK1,NbMEKK2,and NbMAPKK could significantly reduce cell death triggered INF1.This indicated that cell death triggered by Rip25 is independent of pathways mediated by those immune related genes.b)To further investigate function of Rip25,its subcellular localization in plant cells was examined in N.benthamiana by using confocal microscopy.The results showed Rip25(C135S)localized in cytoplasm and nucleus.Interestingly,we observed plethora autophagosome during expression of active form Rip25.The immunoblots also showed autophagic flux occurred during Rip25 expression instead of catalytic mutant Rip25(C135S).Silencing autophagy related gene ATG5 and ATG18 a could not alter cell death triggered by Rip25.This indicated cell death triggered by Rip25 is PCD with autophagy instead of by autophagy.Using three screening systems we identified a highly virulent effector Rip25.Biochemical study revealed that Rip25 is a cysteine protease with ankyrin repeat.Rip25 showed the auto-cleavage during expression in eukaryotic system and the first5 ankyrin repeats is involved in recognition of cleavage site.The cell death triggered by Rip25 are independent of immune related pathway,whereas the autophagy is involved in cell death triggered by Rip25.The Rip25 effector family members existed extensively in plant and animal pathogen which suggested Rip25 protease family are functionally essential for pathogenies in nature.We first report this protease biochemical function in plant and it will enhance understanding of this type of cysteine protease.