Identification of seven Xanthomonas oryzae pv. oryzicola genes potentially involved in pathogenesis in rice

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) in rice, an emerging and destructive disease worldwide. Identification of key virulence factors is a prerequisite for understanding the pathogenesis of Xoc. In this study, a Tn5-tagged mutant library of Xoc strain RS105 was screened on rice, and 27 Tn5 mutants were identified that were either non-pathogenic or showed reduced virulence in rice. Fourteen of the non-pathogenic mutants were also unable to elicit the hypersensitive response (HR) in tobacco and were designated Pth(-)/HR(-) mutants; 13 mutants showed attenuated virulence and were able to induce an HR (Vir(-)/HR(+)). Sequence analysis of the Tn5-tagged genes indicated that the 14 Pth(-)/HR(-) mutants included mutations in hrcC, hrcT, hrcV, hpaP, hrcQ, hrpF, hrpG and hrpX. The 13 Vir(-)/HR(+) mutants included tal-C10c-like (a transcriptional activator-like TAL effector), rpfC (regulator of pathogenicity factors), oxyR (oxidative stress transcriptional regulator), dsbC (disulfide isomerase), opgH (glucan biosynthesis glucosyltransferase H), rfbA (glucose-1-phosphate thymidylyltransferase), amtR (aminotransferase), purF (amidophosphoribosyltransferase), thrC (threonine synthase), trpA (tryptophan synthase alpha subunit) and three genes encoding hypothetical proteins (Xoryp_02235, Xoryp_00885 and Xoryp_22910). Collectively, the 27 Tn5 insertions are located in 21 different open reading frames. Bacterial growth and in planta virulence assays demonstrated that opgH, purF, thrC, trpA, Xoryp_02235, Xoryp_00885 and Xoryp_22910 are candidate virulence genes involved in Xoc pathogenesis. Reduced virulence in 13 mutants was restored to wild-type levels when the cognate gene was introduced in trans. Expression profiles demonstrated that the seven candidate virulence genes were significantly induced in planta, although their roles in Xoc pathogenesis remain unclear.