Horizontal gene transfer (HGT) has been well documented as a driving force in the evolution of bacteria.  It has been shown that a horizontally acquired gene, xoc_2868, involved in the global response against oxidative stress and pathogenicity of Xanthomonas oryzae pv. oryzicola strain BLS256.  However, as a transcriptional factor (TF), the regulatory mechanism of XOC_2868 has not yet been revealed.  Here, evolutionary analysis suggested XOC_2868 might be co-transferred with its physically proximate downstream genes from a Burkholderiaceae ancestor.  Interestingly, RNA-seq data of wild-type (BLS256) and Δxoc_2868 strains under oxidative stress showed that XOC_2868 did not regulate the expression of its adjacent genes, but remarkably influenced the expression of several genes involved in the extracellular polysaccharide (EPS) production and xanthan biosynthesis.  Chromatin immunoprecipitation-sequence (ChIP-seq) combined with transcriptome analysis revealed that XOC_2868 directly regulates a cydAB operon, encoding two subunits of cytochrome bd oxidase and involved in redox balance.  Consistent with Δxoc_2868 strain, cydA- and cydAB-knockout mutants also showed a higher sensitivity to H₂O₂ along with a reduced bacterial virulence compared with the wild-type strain.  Overall, our findings raise the possibility of regulatory circuit evolution shaped by HGT and driven by selection and reveal a novel regulatory pathway that regulates the expression of cytochrome bd oxidase and thus contributes to the virulence of BLS256.  

Bleeding canker (BC) of pear trees, is a devastating disease in China.  The disease was originally observed in Jiangsu Province and its causal agent was identified first as Erwinia sp. in the early 1970’s and latter as a novel species, Dickeya fangzhongdai.  BC is epidemically emerging prevalently from April to September annually in pear-growing regions in Zhejiang, Anhui and Shandong provinces, and threatening pear industry currently in China.  To better control BC disease, it is crucial to know BC symptomatology, epidemics, etiology, and diagnostics first.  The special topic of the three papers have well illustrated the points mentioned above.
The first article by Chen et al. (2020b) described in detail the symptomatology, etiology and epidemiology of BC disease.  BC mainly damages pear trunks and branches with no obvious symptoms early and bacterial rusty oozes mixed with tree saps exuded from died tissues later.  The diseased portions display small soft and sap-filled brown spots or red streaks with strong smells likely of yeast fermentation.  Importantly in this paper, the causal agent is identified as D. fangzhongdai in memory of Prof. Fang Zhongda who was a worldwide distinguished phytopathologist at the Nanjing Agricultural University, China.  The pathogen infects not only pear, but also potato, cabbage, tomato and butterfly orchid, and its virulence is stronger than D.?solani on potato, implying the quarantine is necessary.
Since early symptoms of BC are inconspicuous and the isolation of the pathogen from infected trees is time-consuming, the second paper (Tian et al. 2020) developed a TaqMan real-time PCR technique to determine D. fangzhongdai using specific primers basing on an elongation factor G (fusA) gene of the pathogen.  Efficiently, 0.2 pg µL^–1 DNA and 1×10³ CFU mL^–1 of the bacterium on asymptomatic trees can be detected by this technique, facilitating early and accurate diagnosis of BC five days before visible symptoms appear.
Species in Dickeya genus are originally separated from Pectobacterium and Brenneria.  Some Dickeya speices not only cause diseases in herbal plants, but also exist naturally in lakes or rivers.  To distinguish D. fangzhongdai from other Dickeya speices, the third article (Chen et al. 2020a) reported the genome sequences of three isolates of D. fangzhongdai and compared them with other Dickeya genomes available in NCBI database.  Phylogenetic analysis showed three D. fangzhongdai strains are clustered in one branch, obviously different from other seven Dickeya speices.  Genetic differences are observed in D. fangzhongdai strains in some pathogenicity factors, like type III secretion system (T3SS), T3SS-secreted effector (T3SE), type IV secretion system (T4SS), type IV pili (TFPs), plant cell wall degradation enzymes (PCWDE), and membrane transport proteins, which make the pathogen unique.  Interestingly, the TFP deletion makes D. fangzhongdai lose twitching motility and reduce biofilm formation and virulence in pear.
The findings in the papers enrich our knowledge on how epidemic is the BC disease, what is the pathogen, what tools can be used for BC diagnosis, what are pathogenic and phylogenetic differences of the pathogen from other Dickeya species which can be employed for further quarantine detection.  Believably, the D. fangzhongdai–pear pathosystem set in the reports provides a platform not only for understanding bacterial pathogenicity in the host, but also for developing new technical tools to control BC disease in agriculture. 

Xanthomonas oryzea pv. oryzae (Xoo) is the causal agent of bacterial blight of rice, which is a significant threat to many of rice-growing regions. The type III secretion system (T3SS) is an essential virulence factor in Xoo. Expression of the T3SS is often induced in the host environment or in hrp-inducing medium but is repressed in nutrient-rich medium. The elucidation of molecular mechanism underlying induction of T3SS genes expression is a very important step to lift the veil on global virulence regulation network in Xoo. Thus, an efficient and reliable genetic tool system is required for detection of the T3SS proteins. In this study, we constructed a protein expression vector pH3-flag based on the backbone of pHM1, a most widely used vector in Xoo strains, especially a model strain PXO99^A. This vector contains a synthesized MCS-FLAG cassette that consists of a multiple cloning site (MCS), containing a modified pUC18 polylinker, and Flag as a C-terminal tag. The cassette is flanked by transcriptional terminators to eliminate interference of external transcription enabling detection of accurate protein expression. We evaluated the potential of this expression vector as T3SS proteins detection system and demonstrated it is applicable in the study of T3SS genes expression regulation in Xoo. This improved expression system could be very effectively used as a molecular tool in understanding some virulence genes expression and regulation in Xoo and other Xanthomonas spp.

Bacterial leaf blight (BLB) and bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc), respectively, lead to severe loss in yields and quality in most rice-growing areas around the world. As the staple crop and the model plant for biology research, characterizing the diseases of rice has great significance. It is crucial to elucidate the interaction between the pathogens and rice as well as the mechanisms of pathogenesis and resistance. The special topic of the three papers is the pathogenicity of plant pathogenic bacteria and host resistance to pathogens. 

Transcription activator-like effectors (TALEs) secreted into plant cell through the type III secretion system (T3SS) are important virulence factors of the pathogens. TALEs’ function depends on the direct interaction between its TFB domain and host plant basal transcription factor IIA gamma subunit (TFIIAγ). 

The first article from Tian et al. (2019) investigated the key domain and residues of plant TFIIAγ and core residues of TALE TFB domain that are indispensable for TFIIAγ-TALEs interaction in the process of TALE-carrying Xanthomonas invasion plants, which provides theoretical guidance for the subsequent cultivation of disease-resistant rice varieties. 

The elucidation of molecular mechanism underlying induction of T3SS gene expression is a very important step to lift the veil on global virulence regulation network in Xoo. Thus, in the second paper, Xu et al. (2019) constructed an effective T3SS protein expression vector pH3-flag based on the backbone of almost widely used vector in the pathogens. The experimental results suggested that the expression system will work as a useful molecular tool in understanding some virulence genes expression and regulation in Xoo and other Xanthomonas spp. 

β-Glucosidase responds to various biotic and abiotic stresses, but its function in disease resistance remains unclear. The third article from Li et al. (2019) mentioned that OsBGLU19 and OsBGLU23, encoding β-glucosidases, mediate rice resistance to BLS. They confirmed that β- glucosidase is involved in the interaction between rice and Xanthomonas. 

The findings of the papers above highlight our understanding through several novel perspectives, as well as the operational system, on the interaction between plants and pathogenic Xanthomonas for our goals of plant resistance breeding.

Plant-pathogenic Xanthomonas infects a wide variety of host plants and causes many devastating diseases on crops.  Transcription activator-like effectors (TALEs) are delivered by a type III secretion system (T3SS) of Xanthomonas into plant nuclei to directly bind specific DNA sequences (TAL effector-binding elements, EBEs) on either strand of host target genes with an unique modular DNA-binding domain and to bidirectionally drive host gene transcription.  The target genes in plants consist of host susceptibility (S) genes promoting disease (ETS) and resistance (R) genes triggering defense (ETI).  Here we generally summarized the discovery of TALEs in Xanthomonas species, their functions in bacterial pathogenicity in plants and their target genes in different host plants, and then focused on the newly revealed modes of protein action in triggering or suppressing plant defense.

Carbamoyl-phosphate synthase plays a vital role in the carbon and nitrogen metabolism cycles. In Xanthomonas citri subsp. citri, carA and carB encode the small and large subunits of carbamoyl-phosphate synthase, respectively. The deletion mutation of the coding regions revealed that carA did not affect any of the phenotypes, while carB played multiple roles in pathogenicity. The deletion of carB rendered the loss of pathogenicity in host plants and the ability to induce a hypersensitive reaction in the non-hosts. Quantitative reverse transcription-PCR assays indicated that 11 hrp genes coding the type III secretion system were suppressed when interacting with citrus plants. The mutation in carB also affected bacterial utilization of several carbon and nitrogen resources in minimal medium MMX and extracellular enzyme activities. These data demonstrated that only the large subunit of carbamoyl-phosphate synthase was essential for canker development by X. citri subsp. citri.

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak, a devastating disease in rice-growing regions worldwide. A Tn5-insertion mutant in Xoc_3248, encoding an inner membrane protein (Imp), showed reduced virulence in rice. To explore the potential function of this gene in virulence, a deletion mutant RΔimp was constructed in the wild-type RS105. The RΔimp mutant was significantly impaired for bacterial virulence and growth in planta. The mutation in imp made the pathogen insufficiently utilize glucose, fructose, mannose or pyruvate as a sole carbon source, leading to less extracellular polysaccharide (EPS) production and reduced motility. The deficiencies noted for the mutant were restored to wild-type levels when imp was introduced in trans. Transcription of imp was significantly declined when hrpG and hrpX was mutated and the expression of hrpG and hrpX was also significantly declined when imp was deleted. Cell sublocalization in planta showed Imp membrane-binding feature. These results suggest that Imp is a virulence factor with roles in the catabolism of sugars, EPS production, and bacterial motility.

Xanthomonas oryzae pv. oryzicola (Xoc) causes a destructive bacterial leaf streak disease in rice. Some of the gene products annotated as hypothetical proteins in the genome of Xoc may contribute to its virulence in rice. A mutant, Mxoc1679, screened from our previous Tn5-tagged mutant library for Xoc strain RS105, showed reduced virulence in rice. In this mutant, a gene named as Xoryp_08180 was disrupted by Tn5 insertion. Xoryp_08180 encodes a 1 306-aa hypothetical protein which is highly conserved in Xanthomonas spp. Non-polar mutation of Xoryp_08180 in RS105 strain led to a significant reduction in bacterial virulence and growth in rice, a delayed hypersensitive response (HR) in non-host tobacco, and a decrease in extracellular protease activity. The deficiencies above were restored to wild-type level in the complementary strain by expressing Xoryp_08180 in trans. In addition, the expression of Xoryp_08180 was repressed in hrpG and hrpX mutants in planta but not in a nutrient-rich condition. These results suggested that Xoryp_08180 is a virulence factor required for extracellular protease production, HR induction and full virulence of Xoc.

Xanthomonas oryzae pv. oryzicola (Xoc), the critical pathogen causing bacterial leaf streak in rice, possesses a hrp clusterthat is responsible for triggering hypersensitive response (HR) in non-host tobacco and pathogenicity in host rice, and isconsidered to be one of the model pathogens in the rice model plant. Here, we developed a high-throughput mutagenesissystem using a two-step integration mediated by a novel suicide vector pKMS1. It was used to generate single or poly-genemutants of hpa1, hpa2, hrcV, hrpE, hpaB, and hrpF gene for functional analysis. In total, five single, four double, and twotriple hrp gene mutants were constructed. The double and triple hrp gene deletion mutants triggered novel phenotypesin planta. Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.