Functional Study Of Two Novel E3 Ubiquitin Ligase (Nel) Effectors In Ralstonia Solanacearum

Potato(Solanum tuberosum L.)is the most principal tuber crop worldwide.Potato not only provides food but also increases employment opportunities and economic incomes for mankind.Ralstonia solanacearum ranks second in the top 10 phytopathogenic bacteria and causes bacterial wilt in host plants.Bacterial wilt has caused serious damages to potato production,brought enormous threats to food security and massive losses to national economy.R.solanacearum can translocate type Ⅲeffectors(T3Es)into plant cells through a needle apparatus and channel named type Ⅲsecretion system(T3SS).T3 Es are the main and the most important pathogenicity factors which are crucial to the virulence function of R.solanacearum.R.solanacearum is composed of R.solanacearum species complex(RSSC)and possesses a variety of T3 Es.The diverse functions of T3 Es provide important virulence bases for R.solanacearum on its wide adaptability and strong aggressiveness.In this study,we focus on characterizing the biochemical functions and molecular mechanisms of the two T3 Es RipV1 and RipV2 in the pathogenesis of R.solanacearum.The main results are as follows:1.The enzymatic activities of RipV1 and RipV2Both RipV1 and RipV2 have a conserved Novel E3 ubiquitin Ligase(NEL)domain.Further analysis revealed that both RipV1 and RipV2 belong to the atypical Ipa H(Invasive plasmid antigen H)family effectors,which contain the NEL domain in the C-terminus but no Leucine-Rich Repeat(LRR)domain in the N-terminus.Prokaryotic protein purification and in vitro enzyme activity assay showed that RipV1 and RipV2 exhibited E3 ubiquitin ligase activity which was dependent on the conserved cysteine sites in their NEL domains respectively.2.Subcellular localization of RipV1 and RipV2Subcellular localization analyses in N.benthamiana showed that RipV1 and RipV2 were localized in the plasma membrane(PM),and sequence analyses revealed that neither of them contained transmembrane domains.Subcellular localization analyses of the truncations and mutant showed that the N-terminus was required for RipV1 to be localized in the PM.Further truncation localizations revealed that 1-362 amino acids at the N-terminus played an important role in the PM localization of RipV1.RipV2 was also observed to be localized in the PM.The N-terminal truncation and the NEL domain deletion both conferred a non-unique PM localization for RipV2,indicating that the two parts may be both important for its PM localization.3.Effects of RipV1 and RipV2 on plant immune responsesWhen transiently expressed in Nicotiana benthamiana,RipV1 and RipV2 could both cause cell death on N.benthamiana leaves.However,such cell death would be abolished when the conserved catalytic residue cysteine was mutated.Transient expression of RipV1 and RipV2 could both suppress the pathogen-associated molecular pattern(PAMP)-triggered transcript levels of some PAMP-triggered immunity(PTI)related genes and reactive oxygen species(ROS)bursts.When the corresponding cysteine residue was mutated,all the inhibitions were abolished.These results indicated that both RipV1 and RipV2 were dependent on their catalytic activities to induce cell death on N.benthamiana leaves and suppress plant PTI immune responses.Further genetic transformation assays showed that the transgenic lines overexpressing RipV1 in potato were more susceptible when inoculated with R.solanacearum,while the transgenic lines overexpressing the catalytic mutant exhibited no significant difference compared to the control,indicating that RipV1 is a virulent protein to host plants and the catalytic activity is necessary for this virulence.4.Contributions of RipV1 and RipV2 to the virulence of R.solanacearumBoth the single mutant of RipV1 and RipV2 could cause a significant decrease in the virulence of R.solanacearum UW551,and the reduced virulence could be restored to the level of wild-type UW551 through re-transferred with RipV1 and RipV2 respectively.Therefore,R.solanacearum requires effectors RipV1 and RipV2 for its full virulence and to promote its infection into host plants.5.The interacting proteins of RipV1 and RipV2RipV1 and RipV2 are T3 Es functioning as E3 ubiquitin ligases.The ubiquitinproteasome system not only requires the specific E3 s but also requires ubiquitin conjugation enzymes(UBCs,E2)to transfer ubiquitin.Analyses of the interactions between potato UBCs with RipV1 or RipV2 by yeast two-hybrid showed that RipV1 could only interact with St UBC53,while RipV2 could interact with St UBC23,St UBC51,and St UBC53 among the successfully amplified 49 St UBCs.These results indicate that there may be common E2 and substrate proteins for RipV1 and RipV2 in potato.These provided a basis for further finding out how RipV1 and RipV2 participate in the ubiquitin-protein degrading system in plants.We have screened out some potato interacting proteins of RipV1 by yeast library screening.RipV1 has interactions with six genes encoding Sn RK1β in potato and has bimolecular fluorescence complementation(Bi FC)interactions with four of them.Sn RK1β is an important subunit of Sn RK1(Sucrose Nonfermenting 1 Related Kinase1),which is involved in energy metabolism and regulatory response to stress stimulation in plants.These results suggested that RipV1 may interfere with plant immune responses by participating in energy metabolism in plants via targeting Sn RK1.RipV1 also interacts with St CAP1(CDPK adapter protein 1),an adapter protein of CDPK(Calcium-dependent protein kinase)which plays an important role in the calcium signaling pathway,suggesting that RipV1 may participate in the calcium signaling pathway to interfere with the physiological and biochemical activities of plants.In this study,we characterized firstly the biochemical functions and molecular mechanisms of two NEL domain-containing T3 Es RipV1 and RipV2 in R.solanacearum.We found that these two T3 Es exhibited E3 ubiquitin ligase activities and could suppress the immune responses in plants through their enzyme activities respectively.This study provided theoretical supports for the comprehensive analysis of the virulence factors of R.solanacearum and laid a solid foundation for the in-depth understanding of the interactions between R.solanacearum and potato.