Table_7_Comparative Genomic Analysis of Xanthomonas campestris pv. campestris Isolates BJSJQ20200612 and GSXT20191014 Provides Novel Insights Into Their Genetic Variability and Virulence.XLSX

Black rot is a disease that has a severe impact on cabbage yield and quality in China. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot of Brassicaceae crops. So far, the whole genomic sequences of more than 30 Xcc isolates have been sequenced; however, little information about genomic variability and virulence has been reported. In this study, 12 Xcc isolates were isolated from diseased cabbage leaves in seven Chinese provinces and two municipalities from July 2019 to November 2020. Pathogenicity analysis showed that isolate GSXT20191014 was more aggressive than BJSJQ20200612 and HRIW 3811 on cabbage inbred line 1371. Both BJSJQ20200612 and GSXT20191014 were sequenced and comparatively analyzed. The results showed that BJSJQ20200612 and GSXT20191014 have a single circular chromosome comprising 5,115,975 and 4,975,682 bp, respectively. Compared to the other six sequenced strains, 26 and 47 variable genomic regions were found in BJSJQ2020061 and GSXT20191014 genomic sequences, respectively. The variable genomic regions could be responsible for the genetic variation in Xcc strains and have led to the differences in type III secreted effector repertoires, virulence factors and secreted proteins between these two strains. Among the identified secreted proteins, two copies of peptidase S8/S53 were found in GSXT20191014-specific chromosomal segments. The common effectors xopR, xopH, avrBs1, and xopAH are found in most Xcc genomes, but they are absent in the GSXT20191014 genome. Variations in the composition of exopolysaccharides (EPS) and lipopolysaccharides (LPS) may aid GSXT20191014 isolate infections to evade recognition by the host immune system. Our results revealed a direct correlation between genomic variability and Xcc virulence. We also developed several markers for detecting BJSJQ20200612 and GSXT20191014 isolates and further tested the rest of our other 10 isolates. Finally, the isolated Xcc strains were classified into three genetic subgroups by specific molecular markers and multilocus sequence typing (MLST) approach. BJSJQ20200612 and GSXT20191014 isolates were also classified into two subgroups of Xcc according to the core-genome-based phylogenetic tree. This study extended our understanding of Xcc genomic features and provided the foundation to further characterize the mechanisms for Xcc virulence and a clue for black rot resistance breeding.

黑腐病是严重影响我国甘蓝产量与品质的病害。野油菜黄单胞菌野油菜致病变种（Xanthomonas campestris pv. campestris, Xcc）是十字花科作物黑腐病的致病菌。截至目前，已有超过30株Xcc分离物完成全基因组测序，但关于其基因组变异与致病力的相关研究报道仍较为匮乏。本研究于2019年7月至2020年11月期间，从我国7个省份及2个直辖市的患病甘蓝叶片中分离得到12株Xcc分离物。致病性分析结果显示，在甘蓝自交系1371上，GSXT20191014的致病力强于BJSJQ20200612与HRIW 3811。本研究对BJSJQ20200612与GSXT20191014两株菌株进行了全基因组测序与比较基因组分析。结果表明，两菌株均含单个环状染色体，基因组大小分别为5115975 bp与4975682 bp。相较于已发表的其他6株测序菌株，BJSJQ20200612与GSXT20191014的基因组中分别存在26个与47个可变基因组区域。上述可变基因组区域可能与Xcc菌株的遗传变异相关，并导致两菌株在III型分泌效应蛋白组、致病因子及分泌蛋白组间产生差异。在鉴定出的分泌蛋白中，GSXT20191014的特异性染色体区段内存在2个S8/S53家族肽酶拷贝。常见效应因子xopR、xopH、avrBs1及xopAH在多数Xcc基因组中均有分布，但GSXT20191014的基因组中缺失上述效应因子。胞外多糖（exopolysaccharides, EPS）与脂多糖（lipopolysaccharides, LPS）组成的差异可能助力GSXT20191014侵染宿主并逃避免疫识别。本研究结果揭示了Xcc基因组变异与致病力之间的直接关联。此外，本研究开发了多款用于检测BJSJQ20200612与GSXT20191014分离物的分子标记，并利用其余10株分离物对该标记进行了验证。最终，通过特异性分子标记与多位点序列分型（multilocus sequence typing, MLST）技术，将本研究分离得到的Xcc菌株划分为3个遗传类群。基于核心基因组构建的系统发育树显示，BJSJQ20200612与GSXT20191014分别隶属于Xcc的两个不同亚群。本研究加深了我们对Xcc基因组特征的认知，为进一步解析Xcc致病机制以及开展黑腐病抗病育种提供了理论基础与研究线索。