σ54 factor (RpoN) plays a crucial role in bacterial motility, virulence, growth, and other biological functions. In our previous study, two homologous σ54 factors, RpoN1 and RpoN2, were identified in Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial leaf blight in rice. However, their functional roles, i.e., whether they exert combined or independent effects, remain unknown. In the current study, rpoN1 or rpoN2 deletion in Xoo significantly disrupted bacterial swimming motility, flagellar assembly, and virulence. Transcriptome analysis led to the identification of 127 overlapping differentially expressed genes (DEGs) regulated by both RpoN1 and RpoN2. Furthermore, GO and KEGG classification demonstrated that these DEGs were highly enriched in flagellar assembly, chemotaxis, and c-di-GMP pathways. Interestingly, ropN1 deletion decreased ropN2 transcription, while rpoN2 deletion did not affect ropN1 transcription. No interaction between the rpoN2 promoter and RpoN1 was detected, suggesting that RpoN1 indirectly regulates rpoN2 transcription. In addition, RpoN1-regulated DEGs were specially enriched in ribosome, carbon, and nitrogen metabolism pathways. Besides, bacterial growth was remarkably repressed in ΔrpoN1 but not in ΔrpoN2. Taken together, this study demonstrates the overlapping and unique regulatory roles of RpoN1 and RpoN2 in motility, virulence, growth and provides new insights into the regulatory mechanism of σ54 factors in Xoo.

The type IV pilus (T4P), a special class of bacterial surface filament, plays crucial roles in surface adhesion, motility, biofilm formation, and virulence in pathogenic bacteria. However, the regulatory mechanism of T4P and its relationship to bacterial virulence are still little understood in Xanthomonas oryzae pv. oryzae (Xoo), the causal pathogen of bacterial blight of rice. Our previous studies showed that the σ 54 factor RpoN2 regulated bacterial virulence on rice in a flagellum-independent manner in Xoo. In this study, both yeast two-hybrid and pull-down assays revealed that RpoN2 directly and specifically interacted with PilRX, a homolog of the response regulator PilR of the two-component system PilS-PilR in the pilus gene cluster. Genomic sequence and reverse transcription PCR (RT-PCR) analysis showed 13 regulons containing 25 genes encoding T4P structural components and putative regulators. A consensus RpoN2binding sequence GGN 10 GC was identified in the promoter sequences of most T4P gene transcriptional units. Electrophoretic mobility shift assays confirmed the direct binding of RpoN2 to the promoter of the major pilin gene pilAX, the inner membrane platform protein gene pilCX, and pilRX. Promoter activity and quantitative RT-PCR assays demonstrated direct and indirect transcriptional regulation by RpoN2 of the T4P genes. In addition, individual deletions of pilAX, pilCX, and pilRX resulted in significantly reduced twitching and swimming motility, biofilm formation, and virulence in rice. Taken together, the findings from the current study suggest that the RpoN2-PilRX regulatory system controls bacterial motility and virulence by regulating T4P gene transcription in Xoo. K E Y W O R D S σ 54 , motility, PilRX, type IV pilus, virulence, Xanthomonas oryzae pv. oryzae | 653 YU et al.

Rice grassy stunt virus (RGSV, Tenuivirus) recently emerged on rice in Vietnam, causing high yield losses during 2006-2009. The genetic diversity of RGSV is poorly documented. In this study, the two genes encoded by each ambisense segment RNA3 and RNA5 of RGSV isolates from six provinces of South Vietnam were sequenced. P3 and Pc3 (RNA3) have unknown function, P5 (RNA5) encodes the putative silencing suppressor, and Pc5 (RNA5) encodes the nucleocapsid protein (N). The sequences of 17 Vietnamese isolates were compared with reference isolates from North and South Philippines. The average nucleotide diversity among the isolates was low. We confirmed a higher variability of RNA3 than RNA5 and Pc3 than P3. No relationships between the genetic diversity and the geographic distribution of RGSV isolates could be ascertained, likely because of the long-distance migration of the insect vector. This data will contribute to a better understanding on the RGSV epidemiology in South Vietnam, a prerequisite for further management of the disease and rice breeding for resistance.