Functional Analysis Of Type 3 Secretion System Effectors NopP And GS6880 In Rhizobia–Robinia Pseudoacacia Symbiosis

Rhizobia-legume symbiotic nitrogen fixation(SNF)system is the most efficiency natural nitrogen fixation system.Robinia pseudoacacia,as a leguminous tree with rapid growth,strong stress resistance and strong nitrogen fixation ability,plays a vital role in soil and water conservation,sand fixation and soil bioremediation,thus it has become an important plantation tree species during ecological remediation in the Loess Plateau of China.The research relating to the rhizobia-R.pseudoacacia SNF system will not only contribute to develop the ecological value of R.pseudoacacia,but also deepen the understanding of the SNF mechanism.The Mesorhizobium amorphae CCNWGS0123(GS0123)strain,which infects R.pseudoacacia specifically,was isolated from the nodules of R.pseudoacacia in our lab.Type 3 secretion system(T3SS),as a tool to secrete virulence effectors by animal and plant pathogens,functions during the infection process.Similar to pathogenic bacteria,rhizobia can inject effector proteins into host cells directly to promote the establishment of SNF system via T3 SS.Previous work has proved that T3SS-I of GS0123,which belongs toα-Rhc I family,was required for the establishment of GS0123-R.pseudoacacia SNF system.On this basis,NopP and GS6880 were identified as the type 3 secretion system effectors(T3Es)of GS0123 by sequence alignment analysis and secretion test.With attempt to explore the role of these two effectors in the establishment of GS0123-R.pseudoacacia symbiosis system,nopP knock-out mutant strain △nopP,GS6880 knock-out mutant strain△GS6880 had been constructed by homologous recombination,then plant experiments were carried out subsequently.In the early stage of infection,the physiological indexes of R.pseudoacacia inoculated with wild-type strain and mutant strains were determined,together with the transcriptomics and phosphoproteomics analysis of the R.pseudoacacia root samples.In the late stage of infection,the nodule traits and plant growth performance were counted.Moreover,the internal morphology of the nodule was observed by paraffin section and the bacteroid morphology was observed by transmission electron microscopy.Meanwhile,these two effectors were used as baits to screen their target proteins using yeast two-hybrid assays,and the results were further verified by fluorescence complementation assays and immunoprecipitation assays.In addition,fluorescent protein labeling was used for subcellular localization of effectors and their targets.The results are as follows:(1)nopP and GS6880 were located in the T3SS cluster of symbiotic plasmid of GS0123,and expression levels were up-regulated under the induction of plant symbiotic signal flavonoids.Secretion assays indicated that NopP and GS0123 were effectors of T3SS-I.The up-regulated results of q RT-PCR showed that the two effectors played roles at the early stage of infection.(2)After inoculated with the △nopP,we found that NopP deficiency decreased concentrations of reactive oxygen species(ROS),activity of pectin esterase and pectin lyase enzyme instantaneously in R.pseudoacacia roots during the early stages of infection.Several plant defense-related genes were down-regulated at 36 hpi.Except for a slight decrease in nitrogenase activity and chlorophyll content,there were no significant changes observed in nodule traits and plant performance.These results indicated that NopP might activate plant defense at the early stage of infection,but have little effect on the infection ability and symbiosis efficiency.In addition,subcellular localization analysis revealed that both NopP and its target,trafficking protein particle complex subunit 13-like protein(TRAPPC13),which was identified by screening a yeast two-hybrid library,were co-localized on the plasma membrane.(3)GS6880 deficiency increased the concentrations of ROS,defense-related hormones salicylic acid and jasmonic acid in the R.pseudoacacia roots during the early stage of infection.While the expression levels of some defense-related genes were up-regulated,and the phosphorylation levels were increased.In tobacco leaves,GS6880 inhibited cell death induced by INF1,but not by Bax.Compared with R.pseudoacacia inoculated with wild-type strain,the nodule number of R.pseudoacacia inoculated with GS6880 knockout mutant(△GS6880)increased,while the volume,dry weight and nitrogenase activity of nodules as well as chlorophyll concentration of leaves decreased significantly.However,the nodules were still effective,and there was no significant difference in the internal morphology of nodules and the morphology of bacteroids in the nitrogen-fixing area.These results suggested that GS6880 participated in surpression of the defense response at the early stage of infection.(4)The CDPK-related kinase 3(CRK3)had been identified as a GS6880 target protein in R.pseudoacacia roots by screening a yeast two-hybrid library,and GS6880 interacted with the C-terminal of CRK3.In addition,GS6880 was localized in the nucleus and plasma membrane,while CRK3 was localized in the nucleus,plasma membrane and chloroplast.They can be co-localized in the nucleus and plasma membrane.Based on transcriptome and phosphoproteome data,15 SNF-related genes with significantly changed expression levels and 4 SNF-related proteins with significantly up-regulated phosphorylation levels associated with GS6880 function were identified in R.pseudoacacia roots.In conclusion,NopP and GS6880,which are T3Es of GS0123,interact with TRAPC13 and CRK3 respectively,and play roles in the early stage of establishment of GS0123-R.pseudoacacia symbiosis system.NopP activates R.pseudoacacia immunity immediately,while GS6880 participates in the inhibition of R.pseudoacacia immunity.