Founction Analysis Of T3SS-2 Of Mesorhizobium Amorphae CCNWGS0123

The construction of symbiont between legume and rhizobium is a very complex and sophisticated progress that involved in a set of genes from both sides,and rhizobium type three secretion systems are one of the genes.The whole genome sequencing data showed that our tested strain Mesorhizobium amorphae CCNWGS0123 harbors two type three secretion systems T3SS-1 and T3SS-2.Previous studies suggested that T3SS-1 played an important role in symbiosis with Robinia pseudoacacia.Then,what about the T3SS-2 ? Does T3SS-2also involve in symbiosis with Robinia pseudoacacia? If so,How does T3SS-2 work in symbiosis? If not,what roles does it play in any other aspects?At the outset,we analyzed the components and arrangements of the two gene clusters,the classical regulation sequences invoved in symbiosis,phylogenetic relationships and the expression of the common structure genes of T3SS-1 and T3SS-2.Secondly,based on the homologous recombination,we constructed rhcN2 gene knocked-out mutant Δ2076,which inactivate the T3SS-2.Thirdly,we inoculated Robinia pseudoacacia.with wild type strain and Δ2076 mutant strain,respectively,which help us to figure out the effects of the T3SS-2in the process of symbiosis,such as host plant phenotypic characteristics and the inner structures of nodules.Additionally,bacterial swimming motility and heavy metal resistence of T3SS-2 were explored.Results of this study were as follows:(1)The T3SS-2 was not a simple copy of T3SS-1.Not only the structure components and gene arrangement of T3SS-2,but also regulation sequences and phylogenetic relationships are different with T3SS-1;(2)Compared with T3SS-1,the inducible expression of T3SS-2 structure genes were not response to the induction of flavonoid(a kind of symbiotic signal molecules)after 12 h and 24 h.However,all structure genes of T3SS-2 showed a significant increase after 48 h;(3)The inoculation experiments revealed that there were no significant differences in number and dry weight of nodules between wild type strain and Δ2076 after inoculated for 30 days.However,after inoculated for 45 days,the leaves of the plants that inoculated with Δ2076 turned into yellow.Although root length and stem height have no differences,while nodule number and nitrogenase activity were remarkably decreased.Additionally,we found obvious differences in nodule paraffin sections and TEM sections between wild type strain and Δ2076;(4)Swimming motility of Δ2076 was not changed.The resistance of Δ2076 to Cu2+slightly decreased.In conclusion,Mesorhizobium amorphae CCNWGS0123 T3SS-2 is also involved in the symbiosis with Robinta pseudoacacia but the mechnism of this is still unkown.We speculate that T3SS-2 may play a part in the later stage in symbiosis and it may relate to nodule pro-senescence.