早在2011年，研究者已对其进行了全基因组测序，并发现此基因组中有超过30%的编码基因是假定基因。另外，水平基因转移(horizontal gene transfer，HGT)已被认为是细菌基因组创新和进化的驱动力之一。前人在Xoc应对氧化应激的分子机制研究中，鉴定到了一个参与BLS256响应氧化胁迫并对毒性有贡献的水平转移基因(xoc_2868)。然而，xoc_2868作为一个转录因子的未知编码基因，其调控机制尚未被揭示。本研究基于BLAST序列比对和系统发育分析，初步判断其下游基因(xoc_2866和xoc_2867)与xoc_2868一样，均可能是BLS256在长期进化过程中通过水平转移从伯克氏菌科(Burkholderiaceae)获得的。为探索xoc_2868在BLS256响应氧胁迫过程中的潜在作用，我们分别对野生型(BLS256)和突变株(Δxoc_2868)在氧胁迫处理后进行了转录组测序。RNA-seq数据分析表明，在氧胁迫条件下，突变株中几个参与胞外多糖(EPS)和黄原胶(xanthan)生物合成基因的表达相较野生型显著下调，但未检测到其下游基因(xoc_2866和xoc_2867)的表达。为进一步鉴定受XOC_2868直接调控的基因，我们在野生型xoc_2868 C端融合了His6标签，并对此重组菌株进行了染色质免疫共沉淀 (ChIP-seq)分析。结合转录组分析发现，XOC_2868直接调控一个编码细胞色素bd氧化酶的两个亚基并参与氧化还原平衡的操纵子(cydAB)。与野生型菌株相比，cydA和cydAB缺失突变菌株与Δxoc_2868菌株一致出现对外源H₂O₂敏感性增强和细菌毒力减弱的表型。综上所述，本研究探讨了一种HGT形成和选择驱动的调控回路进化的可能性，xoc_2868与其两个下游基因可能是作为一个基因簇转移的，但它们在BLS256中各自进化，并在外界选择压下得以保留，XOC_2868通过结合新的调控位点直接调控了细胞色素bd氧化酶表达的通路，通过清除H₂O₂和其他ROS保护细胞免受氧化应激。此外，对胞外多糖和黄原胶合成相关基因的间接激活，也促进了其在宿主体内的定植和传播，从而参与了BLS256的致病力。本研究结果强调了在BLS256进化过程中，HGT现象对其毒力和适应性影响的可能性。

水稻细菌性穗枯病又称水稻细菌性谷枯病，是一种由颖壳伯克氏菌 (Burkholderia glumae) 引起的严重的水稻种传病害，对全球水稻生产和食品安全造成了巨大威胁。由于缺乏对B. glumae在植物宿主中的适应性和发病机制的深入了解，迄今生产上还没有有效的防治措施。水平基因转移 (HGT) 已被证明是原核生物进化的主要驱动力。先前对60个Burkholderia全基因组的比较分析推断，大多数Burkholderia基因在其进化过程中至少经历过一次HGT，并在其菌株分化和致病性决定因素中起着重要作用。在本研究中，我们通过对LMG 2196菌株进行全基因组分析，鉴定到了42个潜在的水平转移基因。其中，一个注释为非核糖体肽合成酶(KS03_RS09665)的基因被确定为候选基因。进一步通过系统进化树的建立，发现该基因仅出现在与植物致病相关的Burkholderia菌属，并且在进化分枝上更接近于假单胞菌(Pseudomonas)中编码丁香肽合成酶(SypA)的sypA基因。为研究该基因在B. glumae致病性中的潜在作用，我们构建了syp基因缺失突变株。表型观察结果表明，sypA基因参与调控了该病菌的游动性、生物膜的形成、类似丁香肽代谢物的合成和致病性等重要生理表型。其中，与野生型菌株接种稻穗相比，sypA突变体接种稻穗后发病指数降低了20%。另外，与野生型菌株相比，sypA缺失突变菌株表现为游动能力显著下降、生物膜形成和类似丁香肽代谢物的合成受到抑制。综上所述，本研究探讨了水平转移基因sypA在颖壳伯克氏菌毒力中的作用。结果表明，sypA基因可能参与了颖壳伯克氏菌毒性物质丁香肽的合成，并且正向调控了其游动性和生物膜的形成，从而参与了颖壳伯克氏菌的致病力。本研究的结果强调了在颖壳伯克氏菌进化过程中，HGT现象对其毒力和适应性影响的可能性。

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.