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.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/114038 |
| Date | 03 March 2023 |
| Creators | Li, Qi |
| Contributors | Horticulture, Zhao, Bingyu, Li, Jianyong, McDowell, John M., Bargmann, Bastiaan |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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