Cloning Of TALI_XOC Gene Of Xanthomonas Oryzae Pv.Oryzicola And Inhibitory Effect On Rice Disease Resistance

Bacterial leaf streak(BLS)infection by Xanthomonas oryzae pv.oryzicola(Xoc)has caused serious damage to rice production and attracted extensive attention from the scientific and social community.Xanthomonas secretes toxic or non-toxic factors into host cells through the type Ⅲ secretion systems(T3SS),triggering disease resistance or susceptibility responses in host plants.Among them,transcription activator-like effectors(TALEs)have specific target genes in host plants,which affect the pathogenicity or host disease resistance by regulating target gene expression.Studies have shown that the TALEs of Xanthomonas oryzae pv.oryzae(Xoo)induced the expression of the rice susceptible gene SWEET to produce SWEET protein,which promoted intracellular sucrose transport to the extracellular body to provide nutrients for the pathogen and thus played a toxic role,but this pathogenic mechanism has not been found in Xoc.In this laboratory,researchers inserted Tn5 transposon into the genomic DNA of GD41,a virulence strain of Xoc,and obtained the mutant library of GD41.Through the pathogenicity test,a mutant strain GD21 with significantly decreased virulence was screened out.Southern blot hybridization was performed on the enzyme digestion products of GD21 genomic DNA with the probe labeled Tn5 vector sequence to confirm the single copy insertion of Tn5 transposon.Nucleic acid sequences with TALE gene characteristics were obtained by Tail-PCR,indicating that Tn5 transposon was inserted into a TALE gene.Southern blot hybridization was performed with the conservative TALE gene pthxol as the probe,and Tn5 transposon was inserted into a TALE gene with SphI enzyme digestion fragment size of 2000-bp,which was called Sph2k.As the central region of TALE gene is composed of multiple highly conserved repeat sequences,Sph2k gene cannot be obtained by PCR amplification.In our laboratory,114 positive clones containing TALE gene were screened from GD41 genomic library by in situ hybridization.The positive plasmid DNA was digested by restriction enzyme SphI and Southern blot was performed with pthxo1 gene probe.Four clones containing Sph2k gene were identified as E4,E8,E52 and A57,respectively.Plasmid DNA of E4,E8,E52,A57 and the vector pENTR4-mcs19 were digested by BamHI enzyme,and then transformed into escherichia coli.In situ hybridization was performed with pthxo1 gene probe,and positive clones containing TALE gene were screened.Sph2k gene was obtained by cloning plasmid DNA with BamHI and electrophoresis.Using the Tn5 transposon to randomly insert the Sph2k gene,a number of plasmid mutants with different insertion positions were obtained.The transposon sequencing primer Tn5F and Tn5R were used to sequence these plasmid mutants,and the nucleic acid sequence of Sph2k gene was obtained.Meanwhile,according to the conservative sequence of TALE gene,the author designed two degenerate primers Tail-tal F and Tail-tal R,combined with Tn5 transposon primers,and PCR amplification was carried out using the genomic DNA of mutant strain as the template,And the nucleic acid sequence of Sph2k gene was obtained by sequencing the amplification results.Comparing the two sequencing results,the obtained sequences were exactly the same.PCR amplification with degenerate primers and Tn5 transposon primers provided a simpler and more efficient method for the sequencing of TALE gene.Sequence analysis showed that the central repeat region of Sph2k amino acid sequence was composed of 14.5 repeat units,the gene was named TALIXoc.According to the central repeat sequence of TALIXoc,the Functal program was used to construct an evolutionary tree based on DNA binding specificity.The 12th and 13th amino acid residues(RVD)of the TALE gene center repeat unit determine its binding specificity.The authors predicted the possible target genes based on the TALIXoc RVD sequence on the TAL Effector Nucleotide Targeter 2.0 website.To analyze the mechanism of TALIXoc,the TALIXoc gene Sphl fragment was ligated into the TALE gene universal backbone pZW,and then ligated to the expression vector pHMI by HindⅢ digestion,and the TALIXoc gene expression vector pHZ-TALIXoc was obtained.Studies have shown that the TALE gene of Xoc can inhibit the allergic reaction caused by the interaction between Xoo strain and resistant rice.The TALIXoc gene was introduced into Xoo strain PXO86,and the host rice was treated with PXO86 and PXO86/TALIXoc,respectively.The results showed that TALIXoc gene could inhibit the allergic reaction,callose deposition and oxidative burst induced by PXO86 on host rice.It was demonstrated that the TALIXoc gene can inhibit the innate immune response induced by PXO86 on disease-resistant hosts.TALEs transports into host cells via the type Ⅲsecretory system,activates the expression of target genes and triggers the host’s immune response.Therefore,the transport of TALEs from bacterial cells to rice cells can be detected using the reporting system.Therefore,the transport of TALEs from bacterial cells to rice cells can be detected using the reporting system.Previously,the BlaM reporting system was used to monitor the transport process of PthXol from Xoo cells to rice cells.Therefore,this system was prepared to detect the transport of TALIXoc in Xoc.However,the experiment failed in the process of building BlaM reporting system,so the Cya reporting system was built to detect the transport of TALIXoc.The TALIXoc gene was replenished into the mutant strain GD21 to obtain the replenishing strain GD21/TALIXoc,and its pathogenicity was substantially restored to the wild-type pathogenic level.RT-qPCR analysis showed that TALIXoc significantly inhibited the expression of salicylic acid(SA)defense-related genes,suggesting that the pathogenesis of TALIXoc may be related to the salicylic acid signaling pathway.Specific primers were designed for PCR amplification of the key regulatory genes NPR1 and NPR3,and alternative splicing of NPR3 transcripts was found.However,the expression levels of NPR1 and NPR3 genes in rice did not change significantly after treatment with different strains,indicating that TALIXoc may interfere with the alternative splicing of NPR3 transcripts by activating the expression of target genes.Professor Dong hansong speculated that the target gene of TALIXoc might be GRP4 based on the function of the predicted target protein.RT-qPCR results showed that TALIXoc can significantly increase the expression level of GRP4 gene.The above experiments indicated that TALIXoc may recognize GRP4 gene promoter sequence and activate GRP4 gene expression to produce GRP4 protein.The GRP4 protein interferes with the alternative splicing of the NPR3 transcript and inactivates it,resulting in the failure of the key regulatory gene NPR1 of the salicylic acid signaling pathway to properly regulate the expression of defense-related genes,thereby inhibiting the disease resistance of rice.