Bacterial spot, caused by Xanthomonas species, is a devastating disease of tomato (Solanum lycopersicum) and pepper (Capsicum annuum) (Schwartz et al., 2015). The recessively inherited resistance, bacterial spot 5 (bs5), in pepper (hereafter referred to as Cabs5) can confer resistance against different Xanthomonas strains (Jones et al., 2002). The Cabs5 resistance is characterized by the absence of disease symptoms, faint chlorosis at the site of infection, and reduced bacterial growth. Remarkably, commercial pepper varieties containing the bs5 allele show durable resistance, effectively impeding hypervirulent strain emergence in agricultural fields (Vallejos et al., 2010).

The CaBs5 gene, together with its paralog CaBs5-like (CaBs5L), has recently been cloned (Sharma et al., 2023; Szabó et al., 2023). CaBs5 encodes a 92 amino acid long protein possessing a cysteine-rich transmembrane (CYSTM) domain, which is implicated in various biotic and abiotic responses. Typically, the CYSTM domain contains conserved residues composed of four consecutive cysteines, followed by two hydrophobic amino acids. A recent study suggested that Cabs5 mediating the resistance against bacterial spot lacks these two conserved leucine residues within the CYSTM domain (Szabó et al., 2023).

Tomatoes and peppers are close relatives in the Solanaceae family and commonly susceptible to Xanthomonas infection. Based on the current findings in pepper, we hypothesized that modifying the ortholog of CaBs5 in tomato could confer resistance against Xanthomonas. Consequently, putative Bs5 (SlBs5) and Bs5L (SlBs5L) were identified in tomato based on homology to CaBs5. Both SlBs5 and SlBs5L were located on chromosome 9 with the same head-to-head orientation as their pepper homologues on chromosome 3 (Figure 1a). Despite short and highly similar amino acid sequences of SlBs5 and SlBs5L (Figure 1b), the conserved synteny and gene order in pepper and tomato genomes allowed the assignment of orthology for Bs5 and Bs5L.

The mechanism by which the double leucine deletion in Cabs5 leads to resistance against Xanthomonas remains elusive (Figure 1b). Yet, this deletion in the conserved CYSTM domain could potentially impair CaBs5's native functionality (Abell and Mullen, 2011). Following this assumption, we postulated that knocking out SlBs5 would produce similar outcomes to Cabs5. We aimed to disrupt both SlBs5 and SlBs5L to prevent possible functional complementation by SlBs5L, given their greater amino acid sequence similarity compared to CaBs5 and CaBs5L (Figure 1b).

We constructed a binary vector for Cas9 and a single-guide RNA (sgRNA) targeting conserved sequences present in both SlBs5 and SlBs5L (Figure 1c). Tomato variety Fla. 8000 was transformed with Agrobacterium. From the progeny of successful transformants, we selected two homozygous lines, Slbs5-1 and Slbs5-2, containing frameshift mutations in both genes (Figure 1c). These mutant lines were self-pollinated or backcrossed to the wild-type parent variety to segregate the T-DNA containing the Cas9-sgRNA cassette.

The resistance of the two selected mutant lines was qualitatively evaluated against Xanthomonas perforans GE485 with dip inoculation assays (Figure 1d). At 21 days post-inoculation, the wild-type leaves were covered by black spots indicative of Xanthomonas infection, while both Slbs5-1 and Slbs5-2 retained green leaves with fewer visible symptoms. These phenotypes remained consistent in inoculations of X. perforans 4B and Xanthomonas gardneri 153 (Figure S1).

Quantitative evaluation of bacterial growth further supported these findings. At 5 days post-infiltration with a low-density bacterial suspension, Slbs5-1 showed significant decreases in Xanthomonas populations compared to wild-type plants (Figure 1e). Such reductions were consistently observed for Slbs5-2 (Figure S2). However, Slbs5-1 could not significantly hinder Pseudomonas population growth.

We additionally examined the growth penalty associated with Slbs5-1 and Slbs5-2 in controlled conditions (Figure 1f). The height of plants was measured at two different time points, but no significant differences were observed between the wild type and the two mutant lines (Figure 1f; Figure S3). This suggested that the resistance to Xanthomonas species comes at no developmental cost in the vegetative stage in the laboratory setting.

Although Cabs5-mediated immunity is subtle, it has shown practical value in commercial pepper cultivation. To examine the commercial potential of Slbs5, field trials were conducted with both Slbs5-1 and Slbs5-2 lines at the Gulf Coast Research and Education Center in Florida, a major state for tomato production. Along with naturally occurring Xanthomonas populations, a two-isolate cocktail of X. perforans race T4 was inoculated in the field to heighten disease pressure. Plants were grown with recommended fertilizers and pest management programs, excluding the use of any bactericides or activators of systemic acquired resistance.

Despite seasonal variations, Slbs5 mutant lines consistently maintained reduced disease symptoms (Figure 1g). Additionally, no developmental defects, such as stunting, were observed in these mutants (Figure 1h). Quantification of disease severity, based on visible symptoms caused by Xanthomonas infection on plant leaf surfaces, revealed higher percentages of Slbs5-2 leaves with reduced disease symptoms than wild-type leaves in all tested seasons (Figure 1i; Figure S4). Notably, the Slbs5-2 mutants demonstrated effective resistance during three periods of elevated disease pressure, Spring 2018, Fall 2019, and Fall 2023.

The marketable yield of fruits is a critical consideration in tomato cultivation. We quantified total marketable yield across five seasonal trials, except for two seasons impacted by a hurricane (Fall 2022) and extremely dry weather (Spring 2023). Throughout all seasons, there was no statistically significant difference in marketable fruit yields between Slbs5-2 and the wild-type plants (Figure 1j; Figure S5). However, during the three periods of increased disease prevalence in Spring 2018, Fall 2019, and Fall 2023 (Figure 1i), the mutants consistently showed a tendency to produce a greater quantity of marketable tomatoes (Figure 1j). This possibly suggests a correlation between Xanthomonas resistance of the mutant lines and improved fruit yields.

Overall, this study shows that a knockout of SlbBs5 and SlBs5L in tomatoes represents a promising strategy to achieve broad-spectrum resistance to bacterial spot disease. Compared to stronger sources of resistance, the resistance mediated by Slbs5 and Slbs5L may be considered subtle. However, our mutant lines consistently led to a reduced population of Xanthomonas in laboratory and field conditions. This decrease in pathogen populations could lessen the likelihood of hypervirulent strain emergence. Furthermore, when these mutants are combined with other sources of downstream resistance genes, they may serve as a prior layer of defence. This initial protection has the potential to diminish the probability of pathogen effectors directly interacting with and overcoming the resistance genes, possibly extending the efficacy of durable resistance in the agricultural field.

细菌斑病由黄单胞菌引起，是番茄 ( Solanum lycopersicum ) 和辣椒 ( Capsicum annuum ) 的毁灭性病害（Schwartz等， 2015 ）。辣椒中的隐性遗传抗性细菌斑点 5 ( bs5 )（以下称为Cabs5 ）可以赋予针对不同黄单胞菌菌株的抗性（Jones等， 2002 ）。 Cabs5抗性的特点是没有疾病症状、感染部位出现轻微褪绿以及细菌生长减少。值得注意的是，含有bs5等位基因的商业辣椒品种表现出持久的抗性，有效地阻止了农田中高毒力菌株的出现（Vallejos等， 2010 ）。

CaBs5基因及其旁系同源CaBs5 样基因 ( CaBs5L ) 最近已被克隆（Sharma等人， 2023 ；Szabó等人， 2023 ）。 CaBs5编码一种 92 个氨基酸长的蛋白质，具有富含半胱氨酸的跨膜 (CYSTM) 结构域，与各种生物和非生物反应有关。通常，CYSTM 结构域包含由四个连续半胱氨酸组成的保守残基，后跟两个疏水氨基酸。最近的一项研究表明，介导细菌斑病抗性的 Cabs5 在 CYSTM 结构域内缺乏这两个保守的亮氨酸残基 (Szabó et al ., 2023 )。

西红柿和辣椒是茄科的近亲，通常容易受到黄单胞菌感染。根据目前在辣椒中的发现，我们假设修改番茄中CaBs5的直系同源物可以赋予对黄单胞菌的抗性。因此，基于与CaBs5的同源性，在番茄中鉴定了推​​定的Bs5 ( SlBs5 ) 和Bs5L ( SlBs5L )。 SlBs5和SlBs5L均位于 9 号染色体上，与 3 号染色体上的辣椒同源物具有相同的头对头方向（图 1a）。尽管 SlBs5 和 SlBs5L 的氨基酸序列短且高度相似（图 1b），但辣椒和番茄基因组中保守的同线性和基因顺序允许为Bs5和Bs5L指定直系同源性。

Cabs5中的双亮氨酸缺失导致对黄单胞菌产生抗性的机制仍然难以捉摸（图 1b）。然而，保守 CYSTM 结构域中的这种删除可能会损害 CaBs5 的天然功能（Abell 和 Mullen， 2011 ）。根据这一假设，我们假设敲除SlBs5将产生与Cabs5类似的结果。我们的目的是破坏 SlBs5 和 SlBs5L，以防止 SlBs5L 可能的功能互补，因为与 CaBs5 和 CaBs5L 相比，它们的氨基酸序列相似性更大（图 1b）。

我们构建了 Cas9 的二元载体和针对SlBs5和SlBs5L中存在的保守序列的单引导 RNA (sgRNA)（图 1c）。用农杆菌转化番茄品种 Fla. 8000。从成功转化体的后代中，我们选择了两个纯合系Slbs5-1和Slbs5-2 ，两个基因均含有移码突变（图 1c）。这些突变系进行自花授粉或与野生型亲本品种回交，以分离含有 Cas9-sgRNA 盒的 T-DNA。

通过浸渍接种测定，对两个选定突变株系的抗性针对穿孔黄单胞菌GE485 进行了定性评估（图 1d）。接种后 21 天，野生型叶子被指示黄单胞菌感染的黑点覆盖，而Slbs5-1和Slbs5-2都保留绿叶，但明显症状较少。这些表型在穿孔X. perforans 4B 和Xanthomonasgardneri 153 的接种中保持一致（图 S1）。

细菌生长的定量评估进一步支持了这些发现。在低密度细菌悬浮液渗透后 5 天，与野生型植物相比， Slbs5-1显示黄单胞菌种群显着减少（图 1e）。 Slbs5-2一致观察到这种减少（图 S2）。然而， Slbs5-1不能显着阻碍假单胞菌种群的增长。

我们还检查了在受控条件下与Slbs5-1和Slbs5-2相关的生长惩罚（图 1f）。在两个不同的时间点测量植物的高度，但野生型和两个突变株系之间没有观察到显着差异（图1f；图S3）。这表明，在实验室环境中，对黄单胞菌属物种的抗性在营养阶段无需付出任何发育代价。

尽管Cabs5介导的免疫作用很​​微妙，但它在商业辣椒种植中已显示出实用价值。为了检验Slbs5的商业潜力，在番茄生产主要州佛罗里达州的墨西哥湾沿岸研究和教育中心对Slbs5-1和Slbs5-2品系进行了田间试验。与自然存在的黄单胞菌种群一起，在田间接种了穿孔黄单胞菌 T4 种的两种分离混合物，以增加疾病压力。使用推荐的肥料和害虫管理计划种植植物，不使用任何杀菌剂或系统获得性抗性激活剂。

尽管存在季节性变化， Slbs5突变株系始终保持疾病症状减少（图 1g）。此外，在这些突变体中没有观察到发育缺陷，例如发育迟缓（图1h）。根据植物叶子表面黄单胞菌感染引起的可见症状，对疾病严重程度进行量化，发现在所有测试季节中，与野生型叶子相比，疾病症状减轻的Slbs5-2叶子的百分比更高（图 1i；图 S4）。值得注意的是， Slbs5-2突变体在疾病压力升高的三个时期（2018年春季、2019年秋季和2023年秋季）表现出有效的抗性。

果实的适销产量是番茄种植中的一个关键考虑因素。我们量化了五个季节性试验的可销售总产量，除了受飓风影响的两个季节（2022 年秋季）和极其干燥的天气（2023 年春季）。在所有季节中， Slbs5-2和野生型植物之间的可销售果实产量没有统计学上的显着差异（图 1j；图 S5）。然而，在2018年春季、2019年秋季和2023年秋季这三个疾病流行增加的时期（图1i），突变体始终表现出生产更多适销番茄的趋势（图1j）。这可能表明突变株系的黄单胞菌抗性与提高的果实产量之间存在相关性。

总的来说，这项研究表明，敲除番茄中的SlbBs5和SlBs5L是实现对细菌性斑点病的广谱抗性的一种有前途的策略。与更强的抗性来源相比， Slbs5和Slbs5L介导的抗性可能被认为是微妙的。然而，我们的突变株系在实验室和现场条件下始终导致黄单胞菌数量减少。病原体数量的减少可能会降低高毒力菌株出现的可能性。此外，当这些突变体与下游抗性基因的其他来源结合时，它们可以作为先前的防御层。这种初始保护有可能降低病原体效应子直接与抗性基因相互作用并克服抗性基因的可能性，从而可能延长农业领域持久抗性的功效。