| Bacterial leaf blight of rice caused by gram-negative plant pathogenic bacteria Xanthomonas oryzae pv.oryzae(Xoo)is a kind of vascular disease and seriously affects the sustained growth of rice yield.OS 198 is the representative strain of race 6 of the Xoo Chinese strain.It shows hypovirulence virulence on IRBB series and local variety of rice.Previous studies have found that the two tal genes tal26.5 and tal20.5 in OS 198 can reduce the virulence of PX99A on rice IRBB8,IRBB14,IRBB214,and IRBB50,respectively,but they are not completely inhibited to the level of OS198.PthXo3-2 is a major virulence gene of OS 198,and it has strong virulence function when it is heterologously expressed,but the virulence was not shown in OS 198.EZ-Tn5TM<KAN-2>Tnp Transposome transposon was used to construct a mutant library of OS 198.400 mutants were inoculated on rice IR24 using leaf tip-clipping method.#48 mutant significantly enhanced the virulence of OS 198 in rice IR24,and named as#48-Tn5.Gene fragment flanking the insertion site of the mutant was amplifild by high-efficient thermal asymmetric interlaced polymerase chain reaction(hiTAIL-PCR)and sequencing.The result of NCBI blast homologue searching of the fragment shows the transponson was inserted into Xanthomonas adhein-like protein(ALP)gene.The subclonging and sequencing analysis showed the 3798 bp fragment of ALP encoded 1266 amino acids.Blast analysis showed that ALP has a high homology as 99%with outer membrane protein of Xanthomonas strain South Korea KACC 10331(GenBank Accession No.AAW74096.1),however,its function is unknown.Southern blot of digested genomic DNA with the probe of Km resistence gene fragment proved that#48-Tn5 strain was inserted by a single-copy transponson.the PCR analysis using primers upstream and downstream of ALP respectively verified Tn5 insertion in ALP and with no second transfer of the transponson associated with flanking sequence happened.Genetic complementation by cloning ALP into the wide host range plasmid pHMl and transferring the recombinant plasmid into the#48-Tn5 restored its phenotype as wildtype in the IR24.The virulence of#48-Tn5 strian was tested on(17 IRBB,7 localand 1 IR)rice cultivars by leaf-clipping inoculation,lesion lengths were measured after 15d.The virulence of#48-Tn5 strain on rice IR24 and IRBB 13 were increased.The results suggest that the ALP gene play a vital role in the virulence of OS198.In this study,ALP gene knock-out mutant was generated from OS 198 based on double crossover homologous recombination.And the cosmid pHMALP containing the ALP coding the sequence were eletroporated into the mutant to construct the complementation strain.The ALP gene knock-out mutants showed significantly increased virulence on rice IR24 and IRBB13.We explored the biological function of the ALP gene of OS 198 in rice plant and non-host Nicotiana benthamiana,the function of ALP gene mutants were explored by measuring the growth rate in NB liquid medium and the ability of callose deposits in Nicotiana benthamiana,qRT-PCR detection revealed that the expression of OsSWEET in ALP gene mutants,cya reporting system was taken to test the efficiency of type three secretion(T3SS)of wild-type OS 198 and ALP gene mutants.The results showed that the growth rates of both Tn5 insertion and knock-out mutant strains in NB medium were significantly increased.The lesion lengths and bacterial population of the mutants in the infection rice leaves were significantly higher than that of the wildtype in IR24 and IRBB 13.And callose deposits caused by mutant significantly reduced than OS 198 in the Nicotiana benthamiana.ALP gene plays a vital role in regulating virulence on rice by influencing the expression of susceptible gene OsSWEET14.The detection of cAMP revealed that the loss of ALP gene function did not change the T3SS function. |
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