The Screening For Symbiotic Genes And Identification Of T3SS Effectors In Soybean Rhizobia

Symbiosis between the Rhizobium and legumes plays a key role in the nitrogen cycle of nature and environmental sustainable development. The effective symbiotic process is activated by subtle molecular dialogue between the legume and rhizobia; subsequently, curled root hair wraps the bacteria, develops into the infection thread; then, elongated infection threads help the bacteria enter the cortical cells that would differentiate into nodule primordium; finally, nodule primordium develops into the nodules and the bacteria in the nodules would differentiate into bacteriods that could fix the atmospheric nitrogen. The symbiotic efficiency effectors exist in the entire symbiotic process.In order to explore the potential factors that influence the symbiosis, the study chose Sinorhizobium fredii CCBAU45436 that was regarded as one of predominant population isolated from the Huang-Huai-Hai region (alkaline soil) in China, as the experimental strain. My labmate Dan Wang built a Tn5-tagged mutant library based on 17 different tags and the mutant library contained 25,500 mutants. This study mainly worked on screening the attenuated-competitive-nodulating mutants with PCR-based methods. After first 15 batches of screening,19 attenuated-competitive-nodulating mutants were obtained; combined with the symbiotic experiment that inoculating mutants individually with G. max JD17, finally, tatA, pyrC, nrdJ and zur mutants were found. Among them, the competitive-nodulating ability of tatA and zur mutants were impaired seriously. pyrC mutant couldn’t enter the nodules and only the ineffective abnormal nodules were formed, no bacteriod existed in its nodules; for the nrdJ mutant, it formed the white spherical nodules. These results indicated that the competitive nodulating ability of these mutants that didn’t show symbiosis-deficiency phenotype or only show infection-deficiency, no bacteriod-differentiation or unstable bacteriod-differentiation phenotype, maybe decreased; it suggested that the competitive nodulating phenomennon may happen during the different stages of symbiosis.T3SS could influence the host range of a variety of rhizobia and part of T3SS mutants could promote the specific rhizobia to gain the symbiotic ability with the legume. In addition, nopA mutant that identified as weak-competitive-nodulating mutant during the PCR screening and NopA was the T3SS extracellular structure component. Until now, large amount of studies related to T3SS components have been done; however, the underlying mechanisms about how the TTEs affect the symbiotic specificity are still unknown. By means of cooperation with bioinformatics groups, ten strains from Sinorhizobium fredii, Sinorhizobium sojae, Sinorhizobium sp. I and Bradyrhizobium diazofficiens were predicted as the candiate TTEs. The first part mainly focuses on experimentally identifying the five candidates from USDA110.Q-RT-PCR results indicated that the gene expression of Bll1636, Bll1877 and Bil1804 were induced by genistein and dependent on transcription regulator Ttsl; the protein secretion experiment showed that these three effectors could secret out of the cells and the secretion depended on the T3SS; hypersenstitive response (HR) experiment showed that the three candidates could translocate into the plant cells. In conclusion, Bll1636, Bll1877 and Bll1804 were named as TTEs. Furthermore, the symbiotic test showed that Bll1636 could negatively influence the symbiosis between S. fredii HH103 and G. max Williams 82. Meanwhile, combined with previous study that nine strains from Sinorhizbium owned different symbiotic specificity (host range), the TTEs of narrow-host-range strains were screened and finally,05684GL00001675 was discovered; 05684GL001675 mutant could help S. sojae CCBAU05684 gain the symbiotic ability with G. max JD17; the gene expression of 05684GL001675 was induced by genistein. All these results could indicate 05684GL001675 may be a TTE that influenced the host range. These novel effectors identified in this study would advance the molecular mechanisms study about how the TTEs influence the symbiotic specificity between the rhizobium and different soybean cultivars.