Bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important diseases in rice. However, little is known about the pathogenicity mechanisms of Xoc. Here we have investigated the function of three HD-GYP domain regulatory proteins in biofilm formation, the synthesis of virulence factors and virulence of Xoc. Deletion of rpfG resulted in altered production of extracellular polysaccharides (EPS), abolished virulence on rice and enhanced biofilm formation, but had little effect on the secretion of proteases and motility. In contrast, mutational analysis showed that the other two HD-GYP domain proteins had no effect on virulence factor synthesis and tested phenotypes. Mutation of rpfG led to up-regulation of the type III secretion system and altered expression of three putative glycosyltransferase genes gumD, pgaC and xagB, which are part of operons directing the synthesis of different extracellular polysaccharides. The pgaABCD and xagABCD operons were greatly up-regulated in the Xoc ΔrpfG mutant, whereas the expression of the gum genes was unaltered or slightly enhanced. The elevated biofilm formation of the Xoc ΔrpfG mutant was dramatically reduced upon deletion of gumD, xagA and xagB, but not when pgaA and pgaC were deleted. Interestingly, only the ΔgumD mutant, among these single gene mutants, exhibits multiple phenotype alterations including reduced biofilm and EPS production and attenuated virulence on rice. These data indicate that RpfG is a global regulator that controls biofilm formation, EPS production and bacterial virulence in Xoc and that both gumD- and xagB-dependent EPS contribute to biofilm formation under different conditions.

XA21, as a pattern recognition receptor in rice, senses the quorum-sensing (QS) signal molecule Ax21 secreted by Xanthomonas oryzae pv. oryzae (Xoo) and mediates hypersensitive response-like immunity against the pathogen. However, for the infection of another pathovar, X. o. pv. oryzicola (Xoc), relatively weak defense responses are observed in XA21-containing rice cultivars. In the present study, we demonstrated that the Xoc Δax21 mutant caused more severe disease symptoms than the wild type in XA21 rice cv. IRBB21, but not in non-Xa21 rice cv. Taipei 309. The substance(s) secreted by the wild-type Xoc strain, but not those by the ax21 mutant triggered host immunity against Xoo PXO99 Δax21 in Xa21 rice. Immunoblot analysis corroborated that Xoc, like Xoo, synthesizes and secretes Ax21. Furthermore, the membrane fusion protein RaxA was demonstrated to be required, but the ATP-binding cassette transporter RaxB was dispensable for Ax21 secretion in Xoc. In addition, we demonstrated that Ax21 functions as a QS signal molecule that regulates biofilm formation in Xoc. However, Ax21 signaling is dispensable for bacterial motility, the production of extracellular polysaccharide and protease secretion in Xoc. Interestingly, the two-component system RaxR/H was involved in the regulation of bacterial motility and the regulation was likely independent on Ax21 signaling in Xoc. Taken together, the results indicated that Ax21 secreted by Xoc might induce plant immunity that plays a significant role in rice defense against the pathogen infection.