Tese de doutoramento, Biologia (Microbiologia), Universidade de Lisboa, Faculdade de Ciências, 2018Xanthomonas campestris belongs to the gamma subdivision of Proteobacteria and is the type species of a genus comprising 28 species of plant pathogenic bacteria, affecting 124 species of monocotyledonous and 268 species of dicotyledonous plants. Identified for the first time in 1895 in the United States of America, this species has undergone several taxonomic reclassifications, and currently comprises pathovars X. campestris pv. campestris (Xcc), X. campestris pv. raphani (Xcr) and X. campestris pv. incanae (Xci), causing distinct diseases in vegetables, ornamentals and spontaneous plants belonging to Brassicaceae family, as well as some vegetable crops belonging to the Solanaceae family. Black rot disease, caused by Xcc, is the most important bacterial disease of Brassicaceae, affecting crops and weeds worldwide. After penetration through hydathodes on leaf margins, bacterial multiplication causes typical V-shaped lesions on leaf margins, followed by darkening of the veins that evolve to necrosis of the affected tissue, ultimately leading to plant death. Short-distance dispersal through plant-to-plant contact, human manipulation, wind, insects, aerosols and irrigation water or rain, associated with long distance dispersal through infected seeds and plantlets following commercial routes across the globe are responsible for the worldwide distribution of this disease. Studies on the interaction of Xcc with B. oleracea have identified several virulence genes, however no resistance genes have been successfully cloned in Brassicaceae crops and there is still a lack of effective black rot disease control measures, which continues to cause severe economic losses worldwide. Despite the growing body of work on this subject, molecular mechanisms of host-pathogen interaction have been mostly inferred using in vitro approaches, resulting in a knowledge gap concerning the in vivo behavior of this pathogen during its interaction with plant hosts. In Portugal, an important centre of Brassicaceae domestication, Xcc has long been identified and Portuguese Xcc strains have already been described as a unique sub-population of this pathogen. In this context, with the goal of bringing new insights on the molecular mechanisms of host/pathogen interaction, a set of 33 X. campestris strains collected in the country was characterized, in terms of pathogenicity, virulence, population structure and phylogenetic diversity. Furthermore, the in planta transcriptomes of the Xcc strains representing the extremes of the virulence spectrum during the infection process on two selected hosts were profiled, in a total of four pathosystems. A high level of phenotypic diversity, supported by phylogenetic data, was found among X. campestris isolates, allowing the identification of Xcc closely related pathovars Xcr and Xci for the first time in Portugal. Moreover, among Xcc isolates, presence of races 4, 6 and 7 was recorded, and two novel races of this pathovar, race 10 and race 11, were also described. Contrastingly, the partial virulence profiles determined by the presence of known virulence genes were highly conserved among the set of X. campestris strains. The integration of Portuguese strains in a global dataset comprising a total of 75 X. campestris strains provided a snapshot of the worldwide X. campestris phylogenetic diversity and population structure, correlating the existing pathovars with three distinct genetic lineages. The identification of an intermediate link between Xcc and Xcr, suggests that these pathovars are more closely related to each other than to Xci. This gradient of genetic relatedness seems to be associated to the host range of each pathovar. While Xci appears only to be pathogenic on ornamental Brassicaceae, Xcc and Xcr have a partially overlapping host range: Xcc affects mostly Brassicaceous hosts, whereas Xcr, while affecting the same hosts, is also pathogenic on Solanaceous hosts. These findings suggest that instead of causing a host shift, the genetic divergence between Xcc and Xcr conferred the latter strains the ability to explore additional hosts, resulting in a broader host range. Although population displayed a major clonal structure, the presence of recombinational events that may have driven the ecological specialization of X. campestris and distinct host ranges was highlighted. Portuguese X. campestris strains provided a significant input of genetic diversity, confirming this region as an important diversification reservoir, most likely taking place through host-pathogen co-evolution. Virulence assessment of Portuguese Xcc strains, based on the percentage on infected leaf area after inoculation, highlighted that virulence was not homogeneous within races and that higher pathogenicity was not necessarily correlated higher virulence. It was then possible to select the extremes of the virulence spectrum – CPBF213, the lowest virulence strain (L-vir), and CPBF278, the highest virulence strain (H-vir). The in vivo transcriptome profiling of those contrastingly virulent strains infecting two cultivars of B. oleracea, using RNA-Seq, allowed establishing that Xcc undergoes transcriptional reprogramming in a host-independent manner, suggesting that virulence is an intrinsic feature of the pathogen. A total of 154 differentially expressed genes (DEGs) were identified between the two strains. The most represented functional category of DEGs was, as expected, ‘pathogenicity and adaptation’, representing 16% of the DEGs, although the complex adaptation of pathogen cells to the host environment was highlighted by the presence of DEGs from various functional categories. Among DEGs, Type III effector coding genes xopE2 and xopD were induced in L-vir strain, while xopAC, xopX and xopR were induced in H-vir strain. In addition to Type III effectors, genes encoding proteins involved in signal transduction systems, transport, detoxification mechanisms and other virulenceassociated processes were found differentially expressed between both strains, highlighting their role in virulence regulation. Overall, low virulence appears to be the combined result of impaired sensory mechanisms, reduced detoxification of reactive oxygen species, decreased motility and higher production of pathogen-associated molecular patterns (PAMPs), accompanied by an overexpression of avirulence proteins and a repression of virulence proteins targeting the hosts’ PAMP-triggered immune responses. Contrastingly, the highly virulent strain showed to be better equipped to escape initial plant defenses, whether avoiding detection, or by the ability to counteract those responses. On the other hand, upon detection, highly virulent pathogens will show a decreased expression of avirulence proteins, making them less recognizable by the hosts Effector-Triggered Immunity mechanisms, and thus able to continue multiplying and cause more severe disease symptoms. Through the study of differential infections in planta, the highly innovative strategy used in this work contributed to disclosure of novel virulence related genes in X. campestris pv. campestris - B. oleracea pathosystem, that will be crucial to further detail the virulence regulation network, and develop new tools for the control of black rot disease
