Transposonal Mutation At A TAL Gene Of Xanthomonas Oryzae Pv. Oryzicola Affects Defense Responses Of Rice

Xanthomonas oryzae pv.oryzae(Xoo)and X.oryzae pv.oryzicola(Xoc)represent an important catogory of plant pathogenic xanthomonds and are major bacterial pathogens of rice,causing bacterial blight and bacterial leaf streak in the plant,respectively.Xanthomonads employ various effector proteins to fulfil the pathogenicity function in corresponding host plants,and transcription activator-like(TAL)effectors(TALEs)regulate expression of certain taget genes and modulate the host transciptoem to control plant responses to the bacterial infection.TALEs of Xoo have been shown to activate host-susceptible genes of the SWEET family and promote the production of SWEET proteins,which facilitate sucrose efflux from rice cells and provide a critical nutrition source for bacterial gowth in plant tissues.Whether a similar mechanism is used by TALEs of Xoc is unclear.The present study was tried to analyze the functional mechanism and potential target of any TALE of Xoc characterized by assesing pathogenicity of previously generated Xoc mutants in comparison with the wild-type(WT)strain.A mutant libraty of Xoc was generated in this lab by inserting the transposon Tn5 into genomic DNA of the bacterial strain GD41,allowing for quick identification of a single mutant numbered as GD21,which displayed highly impired level of pathogenicity on the susceptible rice variety Nipponbare.Compared to the WT strain,the mutant was significantly(P<0.01)compromised in the capacity of multiplication within leaf tissues of Nippobare and in the degree of virulence indicated by the severity of bacterial leaf streak symptom formed on leaves of inoculated Nipponbare leaves.Analysis by thermal asymmetric interlaced(TAIL)polymerase chain reaction(PCR)indicated that Tn5 had inserted into the sequence of a TALE gene present in GD21.Based on Southern blotting of SpHI-digested GD21 DNA with a probe specific to a sequence present in the Tn5 transformation vector and a prob specific to conserved seqence of TALE genes,Tn5 is present as a single copy in the chromosome of GD21,and a 2000-bp sequence resulted from SpHI digestion(SpHI-2kb)is characteristic of partial sequence of aTALE gene.This fragment is thought to be a putative TALE gene,temporarily disgnated as SpHl-2k.Based on BLAST searches,the cloned partial sequence of the so-called Sph2k gene is highly similar to those of 56 TALE genes that have been fully or partly characterized in xanthomonads.Thus,the author changes the name gene Sph2k to TALXoxl and designates as talXocl.As different TALE genes contain highly conserved nucleiotide repeats,which cause difficulties in specific amplification of a single TALE gene by PCR,molecular hybridization was performed to determine the number of TALXocl analogs present in the WT Xoc strain GD41.By bacterial colony in situ hybridization and nucleitide dot hybridization with the SpHI-2kbprobe,totally 114 TALE-containing positive clones was isolated from a previously generated GD41 library,and 4 clones were found to carry SpHI-2kb sequences,which were coded as E4,E8,E52 and E57.Thses sequences will be used in the future to identify the TALXol gene by genomic complementation of the talXol mutation for restoration of the phenotypical and molecular characters tested.To reavel the molecular basis of the pahogenicity impairment in the talxol mutant compared to the WT strain GD41,both strains were tested for their capacities of inducing expression of rice genes known to be inducible following TALE activation.Tested genes included 15 TALE targets,which were predicted or characterized previously,and regulatory genes(NPR1 and NPR3)and defense response genes(PR1a and PR1b)involved in the salicylic acid signaling pathway that regulates disease resistance in a variety of plants.To look at possible reasons behind the pathogenicity repression in talXol,this mutant was compared with GD41 for the abilities to induce expression of TALE-inducible genes in inoculated Nipponbare plants.Based on quantitative real-time reverse transcriptase PCR(RT-qPCR)analysis,rice inoculation with GD41 and talXoxl,respectively,did not make differences in expression levels of TALE target genes(codes 07g06970u,02g43760u,02g34970u,03g37840 and 03g07540).By contrast,expression of the gene coded as 09g20100 was highly repressed and that of genes 03g03034u,04g49194u,01g52130u,06g46500u and 06g37080 were largely enhanced in plants inoculated with the talXocl mutatant compared to the WT strain.In plants inoculated with the mutatant,moreover,expression levels of PR1a and PR1b were increased by more than 40 and 80 times,respectively,but both NPR1 and NPR3 were expressed to similar extents in all plants.Quite surprissing,when expressed in plants inoculated with GD41 bacteria,the NPR3 transcript was severely shortened due to lose of long fragments of sequence between 67 and 284 bp,whereas,the normal and correct transcript of NPR3 was detected intalXocl-inoculated plants.This finding suggests that the functional TALxocl employs a unknown mechanism to intefer with alternative splicing of the NPR3 transcript.Taken together,results detailed above suggest that TALXolis a novel TALE gene that has not been reported previously at least in terms of the functional mechanism preliminarily elucidatedd in this study.When being inoculated to Nipponbare plants,the pathogen with\tThe insertional mutation of TALXoci does not affect or differentially influences the expression of 15 known TALE target genes,repressing one and promoting 5 of the TALE target genes at the transcription level.Moreover,TALXoxl may function through interference with the salicylic acid signaling pathway,which essentially involes induced expression of PR genes as molecular markers.In particular,the pathwayessentially involves the roles of NPR3 as a receptor of salicylic acid in sensing of the signaling phytohormone and in metabolic and functional regulation of NPR1.In relevance to this functional network,NRP1 and/or NPR3 may be the target gene of the assumed TALXoxl,and TALXocl is likely to interfere with alternative splicing of the NPR3 transcript,possibly resulting in disfunction of the protenasome for NPR1 degradation.As timely degraditon of NPR1 is required for its fresh production during the transcriptional regulation of PR gens,the distructive effect of TALXocl on NPR3 transcript provides a mechanism for the TALE to facilitate the pathogenicity by depressing the salicylic acid signaling pathway in rice.Thus,functional mechanisms of TALxocl seem distinct from those used by previously studied TALE proteins,which basically target plant SWEET genes to execute the virulent role.