Identification Of The Biosynthetic Pathway For Small Molecular Nematicide Trans-aconitic Acid And Its Applications In Root-knot Nematodes Management

Being able to interact with an extreme wide range of plant hosts,plant-parasitic nematodes(PPNs)have been a constant threat to global agriculture.Since most of the PPNs infect roots,the underground tissues,these pathogens are thus difficult to be detected.Meloidogyne spp.(root-knot nematodes,RKNs)is the most damaging PPNs species that cannot be controlled using current resources.As a potential nematode pathogen,Bacillus thuringiensis(Bt)employs multiple nematicidal toxins.B.thurigiensis CT-43 is a highly toxic strain that isolated and preserved by our laboratory,although it could confer satisfied biocontrol effect in the field,its active components are poorly identified.During the previous studies of exploring novel nematicides from strain CT-43,an unknown but highly produced compound,named CT-A,was found to be nematicidal against M.incognita.However,CT-A was later identified as a known metabolite trans-aconitic acid(TAA,174.11 Da).TAA is the isomer of cis-aconitic acid(CAA),an intermediate of aconitase(ACO)in the tricarboxylic acid(TCA)cycle,and is a strong competitive inhibitor of ACO.Although plants and microbes have been found to biosynthesize and accumulate TAA for a long time,its biosynthetic origins and the physiological relevance of its activity have remained unclear.Started from the newly identified activity for CT-A(TAA),this research aimed at solving the scientific problems proposed above,and the main results of this study are as follows.1)Confirmed and characterized the nematicidal activity for TAA(CT-A)First,by in vitro bioassay against M.incognita,toxicities and LC50 values of the highly purified CT-A and commercial TAA standard were found to be very similar to each other,confirming CT-A to be TAA on bioactivity level.Then,TAA was found to be much toxic than CAA,and this activity is conferred by the trans-conformation of TAA.Moreover,TAA could also significantly inhibit the hatching efficiency of M.incognita eggs.2)Obtained TAA(CT-A)biosynthetic gene cluster and validated the biosynthetic pathway for the first time This study began to isolate CT-A biosynthetic genes before its structure identification.A full sequence-wide deletion strategy covering any possible compound biosynthesis-related genes on plasmid pCT281(281,231 bp)was firstly adopted,since the presence of pCT281 in strain CT-43 was previously shown to closely relate with CT-A production.In total,we designed 26 independent gene knock-out experiments,involving 57 candidate genes,and successfully deleted 13 genes.However,none of them were determined to be CT-A biosynthesis-related by HPLC analysis.Then,the progress in CT-A structure identification made our research direction clear.By searching isomerous function-related genes on p CT281,we found the CT43_RS29745(1,074 bp)gene,which was the sole target of pCT281 and showed sequence homology to reported isomerase;another hypothetical protein-encoding gene CT43_RS29750(912 bp)that was located 111 bp downstream of the CT43_RS29745 gene also attracted our attention,and we named them TAA biosynthesis-related gene A(tbrA)and tbrB,respectively.By heterologous expression,in-frame deletion and complementation of tbr genes,and operon analysis based on RT-PCR,we demonstrated that an operon consisting of tbrA and tbrB is the TAA biosynthetic cluster of strain CT-43.With in vitro catalytic assay,we demonstrated the aconitate isomerase activity of Tbr A protein;with fluorescence microscopy and Western blot,we firstly determined the subcellular localization of TbrB,and confirmed its function as TAA transporter by analyzing the effects of deleting tbr genes and increasing tbrB copy number on cellular TAA secretion.Together,this study revealed an isomerous TAA biosynthetic pathway mediated by tbr operon.Further,the distribution of the tbr operon across B.cereus and B.thuringiensis strains by bioinformatics analysis indicated a general role for TAA in the bacteria-nematode interactions in soil environments.3)Revealed a positive relationship between TAA content and plant inherent ability in resisting nematodesPrevious result from our lab showed that plant-derived TAA are also toxic to M.incognita.In this study,we firstly measured TAA contents in roots of various plants,and distinguished their inherent abilities in resisting nematodes according to scientific references.As a result,the plant TAA contents closely and positively matched their nematode-resistance,which indicates TAA level to be a potential biochemical marker in resistant crop breeding.4)Proposed plant TAA biosynthesis-related candidate genesSequence alignment between tbr gene and the genome of TAA-producing plants showed no homology.To identify TAA biosynthetic genes in plant,we applied genome-wide association study(GWAS)to analyze TAA content data of 455 leaf samples from a maize association population,and identified two candidates: one hypothetical protein(GRMZM5G874896;chr.2)and one auxin-binding protein 1 precursor(GRMZM2G116204;chr.3)protein-encoding genes.This result needs further validation.5)Demonstrated the application potential of TAA resource in RKNs biocontrolSoil incorporation with maize leaf residues significantly inhibited gall formation by M.incognita but not affected plant growth.Irrigation of tomato roots with high TAA-containing Bt fermentation also reduced the gall number substantially.Moreover,TAA was also found to be active in killing another most damaging nematode species,Heterodera spp.Based on these results,a model for multilayered PPNs control strategies using TAA resource was proposed.