Rhizobium Fabae Promote Rhizobium Etli Nodulation Efficiency Through Quorum Sensing

Quorom sensing(QS)is such a signal transmit system,a luxR-type regulator will be activated when N-acyl-homoserine lactones(AHLs)which products by luxI gene reach an appropriate level.luxR protein could induce specific target genes and as well lead to some changes of phynotype,such as expression of toxic gene,formation of biofilm and produce of antibiotics.There are at least three LuxRI-type regulatory systems in Rhizobium etli CFN42:CinRI,RaiRI and TraRI,and cin system is at the top of rai and tra system.cinR is constitutively expression.As is shown in many reports,quorum sensing plays an important role in competitive nodulation.And there are also environmental factors that would affect symbiotic interaction process and this impact may make sense throungh the regulatory system like QS.For example,the effects of other soil rhizobium which could nodule with bean on the quorum sensing of R.etli is not completely understood.Here we tested whether other soil rhizobium-Rhizobium fabae(R.fabae)affects the nodule-forming symbiosis between R.etli and legume hosts.We collected two soil samples which containing rhizobium from Nanjing University of Science and Technology,Nanjing Botanical Garde.Then four strains isolated from root nodules of bean.According to the 16S rRNA gene sequence analysis,three of them belong to genus Rhizobium fabae,which can produce a lot of AHLs.Surface-sterilized and germinated seeds were then placed in sterilized and unsterilized soil,3weeks later count the number of nodules to study whether soil bacteria including rhizobium have effect on nodule formation of R.etli.As a result,there is no difference between WT and ?cinR in number in sterilized soil,but the number of nodules of WT was more than ?cinR in unsterilized soil.To investigate whether soil rhizobium plays a role on nodule formation of WT and ?cinR,we coinoculated R.etli and R.fabae at 1:1 ratio into autoclaved vermiculite with Fahraeus medium,our data likewise suggest that in single-inoculated treatment,WT was same as AcinR in number,when coinoculated R.etli and R.fabae into autoclaved vermiculite,R.fabae could increase the number of nodules of R.etli CFN42 WT.Since R.fabae can generate a lot of AHLs,we speculated that the increased nodule number of WT observed in this study may have been due to the effect of AHLs synthesised by R.fabae on the QS of R.etli.So we measured the expression of cinI-lacZ,traI-lacZ,raiI-lacZ in WT and AcinR with or without the AHLs of R.fabae.At the same time,miller units of the supernatant of WT,AcinR,R.fabae,WT/R.fabae,?cinR/R.fabae were measured at different time points in PY medium.The expression of cinI and tral in WT and raiI in AcinR were induced by the AHLs of R.fabae.And the AHLs of R.fabae did not significantly induce the expression of cinI and tral in a AcinR background and rail in WT,thus influence the AHLs production in mixed culture.Rhizobium infects plants including many complex stages.Biofilm formation and root hair adherence are two of the important stages.In order to know does R.fabae have any effects on the competitive nodulation of R.etli CFN42 WT,biofilm and root hair aherence experiments were assayed in this study.We can saw from data that WT and AcinR have no difference in CFU when cultured individually.Nevertheless,WT strain exhibit increased competition index compared to the AcinR strain in mixed culture.We can concluded that the AHLs synthesised by R.fabae have effects on the quorum sensing of R.etli and then affect the bioform formation and root hair adherence,finally increase the nodulation competitiveness of R.etli WT.