The subject of this work is to develop multiplex-PCR assay for specific detection of plant pathogenic bacteria of Xanthomonas genus causing bacterial spot of tomato. PCR primers for detection of groups A (X. euvesicatoria), B (X. vesicatoria), C (X. perforans) and D (X. gardneri) were developed based on the DNA sequences obtained by sequencing and from the GenBank database (NCBI). Four primer pairs - Xe_shotgun_104, Xe_shotgun_1819, Xv_atpD_403, Xp_efP_202 were designed and subsequently thoroughly tested and optimized for parallel detection of these bacteria. Specificity of the primers was tested on a large complex of bacterial strains pathogenic to tomato and related crops. Following the protocol described above X. vesicatoria, X. euvesicatoria, X. perforans and X. gardneri can be quickly and reliably identified in a single multiplex-PCR assay.

The thesis deals with methods for viability evaluation of the phytopathogenic bacterium Xanthomonas euvesicatoria after low-temperature plasma treatment. Low-temperature plasma produced by Gliding Arc experimental device was used for treatment of X. euvesicatoria. The viability of the bacterial cells was assessed using a scanning electron microscope (SEM) and by measuring of the fluorescence in the Smart-DART device using PrestoBlue chemical reagent. Methodology has been optimised for the sample preparation for the treatment by low temperature plasma and used for evaluation of applied methods. Lethal effect of gliding arc plasma to this gram-negative bacteria was verified by SEM which showed. There is significant structural changes on the cell surface. Viability assessment of X. euvesicatoria using Smart-DART device is a fast, time-saving and inexpensive evaluation of cell viability. The great advantage of this device is its ability to measure the fluorescence in real time. The disadvantage of this method is lower reliability in current stage of research.

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Pepper is a popular and important vegetable crop grown and consumed worldwide. However, pepper production is threatened by the gram-negative bacterium Xanthomonas euvesicatoria (Xe) which causes bacterial spot (BS) disease, one of the most common and destructive diseases on pepper. Due to limited genetic resistance resources in host species, a promising strategy for controlling BS disease is to transfer nonhost disease resistance (R) genes from other plant species into pepper plants to confer broad-spectrum and durable resistance. A maize R gene Rxo1 has been functionally transferred to rice plants and confers nonhost resistance to rice pathogen Xanthomonas oryzae pv. oryzicola (Xoc) carrying a type III effector (T3E) AvrRxo1. Most Xe strains carry a T3E Xe4428, a homolog of AvrRxo1. Therefore, Rxo1 could be potentially employed to develop Xe-resistant pepper. In addition, a better understanding of the virulence function of Xe4428 may provide insights into the pathogenesis of Xe and new strategies for crop improvement. In this dissertation, we transformed Rxo1 into the far-related dicot species Nicotiana benthamiana and pepper, and characterized the Rxo1-mediated disease resistance against Xe strains carrying AvrRxo1 or Xe4428. In addition, we explored the virulence function and mechanism of Xe4428. In the Rxo1-transgenic N. benthamiana, we demonstrated that Rxo1 could condition resistance to Xe harboring AvrRxo1 but not Xe4428. We revealed that AvrRxo1 could directly interact with the nucleotide-binding domain of Rxo1 in vivo and in vitro. We further demonstrated that the nucleus localization of AvrRxo1 was required for its avirulence and virulence functions. In addition, the cytosol localization of Rxo1 was also necessary to confer disease resistance. The downstream signaling component NbNDR1 was demonstrated to be involved in Rxo1/AvrRxo1-mediated disease resistance. By RNAseq-based gene expression profiling, we identified six candidate genes of interest up-regulated by the Rxo1-AvrRxo1 recognition. Through virus-induced gene silencing screening, a gene encoding phenylalanine ammonia-lyase 4 was demonstrated to be critical for Rxo1/AvrRxo1-mediated disease resistance in N. benthamiana. Rxo1-transgenic pepper plants were resistant to the Xe strain with the complementary Xoc effector AvrRxo1 but not the wild-type Xe strain that carries Xe4428. A Xe4428 mutant with only one nucleotide substitution could trigger the Rxo1-mediated disease resistance in pepper. Both wild-type and mutant Xe4428 had significant virulence functions that could promote the Xe bacterial proliferation on wild-type pepper plants. In addition, the mutant Xe4428 had a higher expression level than wild-type Xe4428 in Xe bacterial cells, which might explain why the mutant Xe4428 but not wild-type Xe4428, could trigger the Rxo1-mediated disease resistance in pepper. We identified 14 pepper cystatin genes (CaCys), among which two genes (CaCys1 and CaCys13) could be induced, and two genes (CaCys3 and CaCys5) were suppressed by Xe4428. Ectopically expressing one of the induced genes CaCys1 in N. benthamiana increased the stomatal opening and promoted the Xe growth in N. benthamiana plants. Thus, we illuminate one possible mechanism of Xe4428's virulence function is to regulate the stomata apertures in N. benthamiana. Bacterial fruit blotch (BFB) caused by the gram-negative bacterial pathogen Acidovorax citrulli (A. citrulli) is one of the most destructive diseases in cucurbit crops, including melon and watermelon. A better understanding of the virulence and avirulence functions of T3Es in A. citrulli helps breeders engineer crop resistance to BFB. To this end, a clean genetic background of A. citrulli with multiple effector genes deleted is desired. Here, we optimized a marker-exchange-based method for sequential effector deletion and generated an AAC00-1 mutant with five effector genes (Aave2166, Aave3626, Aave1548, Aave2938, Aave2708) deleted (AAC00-15). AAC00-15 was less virulent in watermelon but more virulent in N. benthamiana. Through complementation, we characterized the function of individual effectors and identified a promising R gene, Roq1, that could be used to control BFB disease. / Doctor of Philosophy / As an essential ingredient in almost all cuisines, pepper is grown and consumed worldwide, providing human beings with favorable flavor and nutrients. However, pepper production is threatened by the destructive bacterial spot (BS) disease caused by the bacterial pathogen Xanthomonas euvesicatoria (Xe). Due to limited genetic resistance resources in host species, nonhost resistance (R) genes from other plant species are desired to confer broad-spectrum and durable resistance to the pepper pathogen Xe. Previously, a maize (corn) R gene called Rxo1 was transferred to rice plants. This gene helped these rice plants resist a rice bacterial pathogen that causes leaf streak disease on rice. This rice pathogen has an effector (a virulent protein produced by bacteria to infect plants) that is required for the disease resistance. The pepper pathogen carries a similar effector, so transferring the maize R gene Rxo1 to pepper plants might similarly benefit peppers and help fight against the bacterial spot disease. In this dissertation, we successfully transferred the maize R gene Rxo1 into Nicotiana benthamiana and pepper plants. Our results indicate that this gene can help control disease caused by the pepper pathogen harboring the effector of the rice pathogen but not its native effector. We also illuminate how the disease resistance conferred by this maize gene happens in Nicotiana benthamiana plants. In addition, we explain how the corresponding effector helps infect plants. This research provides insights into the application of R gene transfer between far-related plant species and new tools to improve crop disease resistance.

Pepper and tomato, as two common, popular, and important vegetables grown worldwide, provide human beings with high quality fruit of flavor and aroma, and a high concentration of vitamins and antioxidants. Pepper and tomato production is frequently affected by various pathogens, including nematodes, fungi, and bacteria. Among those phytopathogens, Xanthomonas euvesicatoria (Xe) causes a severe bacterial spot (BS) disease on pepper and tomato. The BS disease could cause a loss of approximately 10% of the total crop yield in the world. Breeding tomato and pepper cultivars with improved BS disease resistance is one of the most important breeding goals. A better understanding of the virulence mechanism of Xe could help breeders design new strategies for resistance breeding. In this dissertation, we characterized the virulence and avirulence functions of four Xe Type Three Secretion Effectors (T3Es): Xe-XopQ, Xe-XopX, Xe-XopN, and Xe-avrRxo1. Xe-XopQ is a Xe T3E that functions as a determinant of host specificity. Here, we further explored the virulent and avirulent functions of Xe-XopQ. We identified another T3E Xe-XopX that could interact with XopQ and subsequently elicit the hypersensitive response in N. benthamiana in the Agrobacterium-mediated transient assay and Xe-mediated disease assay. The interaction is confirmed by bimolecular fluorescence complementation, co-immunoprecipitation and split luciferase assay. Intriguingly, we also revealed that XopX also interacts with multiple Xe T3Es including AvrBS2, XopN, XopB, and XopD in the co-IP assay. The virulent and avirulent functions of XopQ and AvrBS2 are compromised in the absence of Xe-XopX. Since XopX is conserved in diverse Xanthomonas spp., we speculate that Xe-XopX may have a general role required for the pathogenesis of Xe. Xe-XopN has been reported to be a T3E with virulence function via targeting host defense-related proteins, including atypical receptor-like kinase named TARK1 and a 14-3-3 protein to suppress the PAMPs (pathogen-associated molecular patterns) triggered immunity upon Xe colonization of tomato. In this study, we revealed additional virulence mechanisms of Xe-XopN, where Xe-XopN, is required for triggering the water-soaking symptom on Nicotiana benthamiana and pepper plants infected with Xe. In addition, we identified that XopN interacts with a transcription factor, NbVOZ, and represses the expression of NPR1, a key component of the basal defense. Therefore, XopN has a role in maintaining a water-affluent environment for better replication of Xe, and it can also interact with NbVOZ1/2 to regulate plant immunity. AvrRxo1, a T3E of Xanthomonas oryzae pv. oryzicola (Xoc), was previously identified to function as a NAD kinase. Here, we characterized a Xe T3E, Xe avrRxo1, that is a functional homologue of AvrRxo1, which is required for the full virulence of Xe to colonize the pepper and N. benthamiana plants. Overexpression of AvrRxo1 in bacterial or plant cells is toxic. Our group previously demonstrated AvrRxo1-ORF2 functions as an antitoxin that binds to AvrRxo1 to suppress its toxicity. In this study, we identified Xe4429 as the homologue of AvrRxo1-ORF2, which could interact with Xe-avrRxo1 to suppress its toxicity. We also revealed that Xe4429 could bind to the promoter of Xe-avrRxo1 and suppress its transcription. Therefore, we found Xe4429 encodes protein functions as an antitoxin and a transcription repressor in Xe bacterial cells. / Doctor of Philosophy / Peppers and tomatoes are two of the most important vegetables grown worldwide, providing humans with high quality of flavor and aroma, vitamins, and antioxidants. The pepper and tomato production is frequently threatened by various pathogens, including nematodes, fungi, and bacteria. Among those phytopathogens, Xanthomonas euvesicatoria (Xe) causes a severe bacterial spot (BS) disease on peppers and tomatoes. The BS disease can be easily identified due to the appearance of the dark, irregular, water-soaked areas on the leaf, which can cause approximately 10% loss of the total yield of peppers and tomatoes. Breeding tomato and pepper cultivars with improved BS disease resistance is one of the most critical breeding goals. A better understanding of the virulence mechanism of Xe could help breeders to design new strategies for resistance breeding. In my seminar, I will discuss the virulence and avirulence functions of Xe type three secretion (T3S) effectors: Xe XopN, Xe XopQ, and Xe XopX. In my study, I identified Xe XopN is a key factor that regulates the development of the water-soaking symptom on pepper plants infected with Xe. In addition, we revealed Xe XopN interacts with a transcription factor NbVOZ to regulate the expression of NbNPR1 and PR1 genes expression, which may also contribute to the development of water-soaking phenotype. In addition, I identified that Xe XopN could interact with a transcription factor, NbVOZ, and represses the expression of NbNPR1, a key component of the basal defense, and the pathogenesis-related gene PR1. Therefore, Xe XopN has a role in regulating a water-affluent environment to promote bacterial proliferation in the infected plant tissue. Xe XopQ is a Xe T3S effector that functions as a determinant of host specificity. In my study, I identified another T3S effector Xe XopX that could interact with Xe XopQ to trigger the defense response in Nicotiana benthamiana. I also confirmed Xe XopQ physically interacts with Xe XopX inside of plant cells by using bimolecular fluorescence complementation, co-immunoprecipitation and split luciferase assay. Intriguingly, Xe XopX could also interact with multiple Xe T3Es including AvrBS2 in a co-IP assay. The virulence and avirulent functions of Xe XopQ and AvrBS2 are compromised in the absence of Xe XopX.

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