Study The Molecular Mechanism Of Rhizobium Etli CFN42 Acquired Symbiosis Island And Evolution In Sesbania Rostrata

Rhizobium symbiotic genes are usually carried on symbiotic islands or plasmids and can be transferred between different rhizobium bacteria.Previous laboratory studies have shown that Azorhizobium caulinodans ORS571 can establish a stable symbiotic relationship with Sesbania rostrata.The symbiosis island of ORS571 can be transferred to other rhizobium bacteria,and the obtained ICE^AC rhizobium can form a symbiotic relationship with Sesbania,and fix N₂ in the air（effective nodules）.Whereas,Rhizobium etli CFN42 cannot receive symbiotic island.CFN42Δ1180 can receive symbiosis island after RHE＿RS01180 was knocked out,but CDZY0 can’t form nodules with Sesbania;Deletion of RHE-RS30755(nodU^CF)gene in CDZY0,the strain CDZY0ΔnodU^CF can form ineffective nodule,but can’t form a symbiotic relationship with Sesbania.This paper studied the mechanism of ICE^AC obtained by CFN42Δ1180.The experimental evolution of CDZY0ΔnodU^CF was carried out using host selection pressure of Sesbania.We obtained mutant strains capable of forming effective nodule.The main research results are as follows:（1）The mechanism of ICE^AC acceptance by CFN42Δ1180 was studied:The transcriptome data were analyzed and the strain were verified by pulse-field electrophoresis.It was found that the large endogenous plasmid p42f was lost in CFN42Δ1180.Plasmid stability related genes（RHE＿RS33905 and RHE＿RS27355）were knocked out under WT（Wild type）background to verify whether the plasmid is involved in inhibiting the acceptance of symbiosis island.The elimination plasmid p42f strain（CFN42ΔTS）was conjugated with ORS571（Azc2）and could not accept ICE^AC.It was proved that plasmid p42f had no effect on the ICE^AC acceptance process of CFN42.Bioinformatics method was used to analyze differentially expressed genes in CFN42Δ1180/WT CFN42,and the 25 candidate genes screened contained conserved sites that could be bound by 1180 protein.Gene expression was analyzed by q RT-PCR,and the results were basically consistent with the results of RNAseq analysis.Seven candidates were further selected,which may influence on the process of ICE^ACacceptance by CFN42.（2）In order to study the evolutionary mechanism of CFN42 in Sesbania,CDZY0ΔnodU^CF was inoculated into the root of Sesbania,isolated from the nodules,and then inoculated again for continuous passage to build a pedigree of adaptive evolution.After the first round of evolution,the 1U1 strain isolated from the largest nodule were obtained as the starting bacteria for subsequent successive passage（the number on the left of U represents the passage times,and the number on the right represents a nodule）.In this paper,7 forward mutants were obtained from the third round of passage.Among them,3U8,3U1 and 3U8 could produce effective nodule after5 rounds of continuous inoculation.The detection of the number of bacteria in the nodule showed that all positive mutants could fill the plant cells inside the nodule,and the number of bacteria in the effective nodule formed was 10 times that of the ineffective nodule.Nitrogenase activity was lower in nodules produced by inoculation with positive mutant 7U than that of inoculation with Azc2,and the number of effective nodules was less,indicating that there were differences in the nitrogen fixation ability between the isolated mutant and the wild-type strain,and there was room for further evolution.When the forward mutant was inoculated into Phaseolus vulgaris（the original host）,it retained the ability of nodulation,and the activity of nitrogenase was higher than CDZY0ΔnodU^CF,indicating that the adaptability to Sesbania and Phaseolus was improved.The host range of positive mutants was detected,and it was found that the positive mutants could produce nodules in soybean hosts,which expanded the host range of symbiosis.（3）The study of key genes that influence adaptive evolution:Propagating map was divided into three evolutionary lines（2U1/3U8,2U2/3U1 and 2U3/3U9）.The effective nodule isolates（3U）and the previous generation of ineffective nodule isolates（2U）and 1U1 were re-sequenced,and the mutant genes were compared and analyzed.The types and number of mutated genes in different evolutionary lines are different,suggesting that mutants used different evolutionary strategies to adapt to host selection pressure.Bioinformatics method was used to analyze the key mutant genes in combination with the transcriptome data,showed that mutations in genes expressing OmpA,NAT and Tolc might be associated with effective nodule formation.We studied the molecular mechanism of directed evolution of CFN42 in Sesbania,and found the relevant proteins and key genes that may affect the evolutionary process,and enhanced the symbiosis ability of the forward mutants in the original host,and realized bidirectional evolution,which initially clarified the evolutionary process and mechanism of CFN42 in Sesbania.