Knock-out of pathogenicity-related genes in Xanthomonas oryzae pv. oryzicola and Xanthomonas oryzae pv.oryzae

wxocA,wxocB,wxocD,wxocE and wzt are genes for lipopolysaccharide(LPS)biosynthesis in Xanthomonas oryzae pv.oryzicola(Xooc),and avrXa3 is an avirulence(avr)gene in Xanthomonas oryzae pv.oryzae(Xoo).Plasmids pBCSK(-)and pKNG101 with replicon of ColE1 and R6K,respectively,were used to construct gene knock-out transformation units.Transformation units of pBCSK∷ΔwxocA,pBCSK∷ΔwxocD,pBCSK∷ΔwxocE and PBCSK∷Δwzt to produce Xooc strain PS105 corresponding gene mutants were constructed with two fragments:central portion of target gene and pBCSK(-) vector.Transformation units of pKNG101∷ΔwxocB and pKNG101∷Δavr to produce wxocB mutant of Xooc strain RS105 and avirulence gene mutant in Xoo strain PXO99 were constructed with four fragments :pKNG101 vector,two flanking sequences of corresponding gene,and cm gene.Transformation was conducted by electroporation to yield 5 lps gene mutants(MwxocA,MwxocB,MwxocD,MwxocE,Mwzt)and 1 avr gene mutant(PXO99Δavr),and homologous recombinations were confirmed by Southern blot.The analysis of LPS by SDS-PAGE showed that all the lps gene mutants had defects in O-antigen biosynthesis.Virulence tests indicated that pathogenicity of MwxocB and MwxocE were completely lost and Mwzt was reduced.And PXO99Δavr showed less virulence on IRBB50(Xa4/xa5)and Asominori(Xa17).Therefore,the vectors pBCSK(-)and pKNG101 were first confirmed to be successfully used to knock-out genes in Xooc and Xoo and be helpful to identify the functions of pathogenicity-related genes in Xooc and Xoo.