Function Analysis Of T3ss Putative Effector 6739 Of Mesorhizobium Amorphae CCNWGS0123 In Symbiosis

The symbiotic nitrogen fixation process of rhizobia and legumes plays a very important role in the natural nitrogen cycle system.Signal exchange between rhizobia and legumes eventually give rise to the formation of nodule,a specific nitrogen fixation organ.The establishment of symbiotic relationship involves complex gene regulation,signal transduction and molecular interactions.Previous researches demonstrated that inactivation of type III secretion systems of Mesorhizobium amorphae CCNWGS0123 significantly inhibited the symbiotic process with Robinia pseudoacacia.Based on the genome sequencing,6739 is one of putative effectors of the type III secretion system.The aim of this study was to demonstrate whether this putative effector would affects the symbiosis with Robinia pseudoacacia and whether 6739 is a T3 SS effector.Firstly,conserved tts box sequence was used to retrieve the promoter region of 6739,and then 6739 was quantified after flavonoids indution in vitro to show whether the expression of 6739 was induced by plant symbiotic signal.Secondly,deletion mutant ?6739 was constructed.the wild type strain and mutant strain were inoculated to Robinia pseudoacacia respectively.The effects of 6739 on the symbiotic process were inferred by observing the plant phenotypes and the internal morphology of root nodules.In addition,6739 was transformed into Agrobacterium for transient expression in tobacco leaves to determine whether 6739 was toxic to tobacco.Finally,6739 was expressed in prokaryotic cells and then purified.The main experimental results are as follows:(1)The tts box-like sequence was found at 222 bp upstream of 6739;(2)qRT-PCR results showed that the relative expression level of 6739 was up-regulated after inducing with flavonoids;(3)The inoculation experiments revealed that the deletion of 6739 gene significantly affect the growth of Robinia pseudoacacia.Compared with the plants inoculated with WT,the plants that inoculated with ?6739 were smaller with decreasing effective nodules while ineffective nodules were increased,and the leaves of the plants turned into yellow.The nodule paraffin sections and TEM sections showed that the internal structure of ?6739 nodules was abnormal;(4)After transient expression of 6739,lesions regions were shown in the tobacco leaves;(5)The 6739 protein with high purity was successfully expressed and purified.In conclusion,type III secretion systems putative effector 6739 of Mesorhizobium amorphae CCNWGS0123 act as a positive factor in symbiosis with Robinia pseudoacacia.However,the exact function of 6739 in symbiotic process and whether 6739 is a T3 SS effector still need further verification.