Bacterial spot of tomato (Solanum lycopersicum) is a devastating disease that severely limits yields in important tomato-­growing regions, including the southeastern United States, where the predominant bacterial spot pathogen species is Xanthomonas perforans. Attempts to control the disease with antibiotics and copper-­based pesticides have led to the selection of bacterial strains that are resistant to these treatments. Therefore, we turn to genetic sources of resistance as a sustainable path to reduce crop losses to bacterial spot disease.

This work describes the fine mapping and identification of the RXopJ4 disease resistance locus from the wild tomato relative Solanum pennellii LA716. RXopJ4 resistance depends on recognition of the X. perforans type III effector protein XopJ4. We developed a collection of fourteen molecular markers to map on a segregating F2 population from a cross between the susceptible parent S. lycopersicum FL8000 and the resistant parent RXopJ4 8000 OC7. In the F2 population, a 190-­kb segment on chromosome 6 cosegregated with resistance. This fine mapping enabled the identification of three RXopJ4 candidate genes, all of which encode putative intracellular serine-­threonine protein kinases. Transient coexpression of the XopJ4 effector with each kinase revealed a promising RXopJ4 candidate gene that triggered a hypersensitive response (HR) in Nicotiana benthamiana. Mutations in both XopJ4 and RXopJ4 identified conserved residues required for recognition and the induction of a hypersensitive cell death phenotype. Homozygous transgenic tomato plants containing the RXopJ4 candidate gene have been constructed and will soon be evaluated for disease resistance.

In addition, we undertook a genomic survey of fourteen X. perforans field isolates from all five fresh market tomato production zones of Florida, revealing a preliminary set of core type III effectors common to all isolates. We used this set of core effectors to inform a search for new sources of resistance to bacterial spot disease and identified accessions of Nicotiana and Solanum americanum that recognize core X. perforans effectors. Finally, we performed disease assays on 224 wild tomato accessions and found nine potential sources of bacterial spot disease resistance.

Cassava is an essential food crop relied on by hundreds of millions of people worldwide. Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of cassava bacterial blight (CBB) and the leading destructive bacterial pathogen of cassava. Xam utilizes a unique class of type three effectors known as transcription activator-like (TAL) effectors (TALEs) to activate specific host genes that contribute to virulence and bacterial growth. In Xam, TALEs are often localized to plasmids. TALE-containing plasmids of other Xanthomonas ssp. have been shown to be conjugative, providing a mechanism for the horizontal transfer of TALE virulence components. Here, I characterize the two-TALE containing plasmid pXam46 of Xam isolate CIO151, providing a full draft sequence, TALE virulence assays, and evidence for its mobilizing ability. The potential horizontal transfer of TALEs suggests that TALEs which confer a strong virulence phenotype may be well conserved amongst Xam communities. I screened a subset of 22 global Xam isolates spanning 28 years of evolution from 5 countries over 3 continents for their TALE repertoires. I identified one pair of highly conserved TALEs, including a single repeat variable diresidue (RVD) variant of a pXam46 localized TALE, and 3 additional well-conserved TALEs. Of the two highly conserved TALEs, both contribute to bacterial growth in planta and one is associated with a water soaking disease phenotype. The remaining 3 well-conserved TALEs did not show any measurable contributions to virulence. Some plants contain an evolutionary mechanism to defend against TALEs, carrying executor resistance (R) genes containing TALE binding elements upstream of disease resistance genes. I found that the highly conserved TALE of pXam46 triggers a specific, transcriptionally dependent HR-like phenotype in the non-host Nicotiana benthamiana. Employing RNA-seq, I have identified a list of candidate TALE-upregulated genes that may be involved in the defense response of N. benthamiana. Identifying TALE-triggered R genes as well as conserved TALEs and their susceptibility targets can assist in the design of durable resistance strategies against Xam. Successful strategies may include stacking promoters of multiple conserved TALEs in front of R genes or modifying cassava susceptibility gene promoters to abrogate TALE binding. It is my hope that the basic biology described herein may assist in those efforts.

Xanthomonas spp are the causative agents of bacterial spot disease on cultivated pepper, Capsicum annuum, and tomato, Solanum lycopersicum. Although pepper and tomato are closely related in the Solanaceae, four species of xanthomonads have differing host specificities and utilize unique virulence strategies between these two crops plants. A major factor differentiating these pathogens are the Type III Effector (T3E) proteins they deploy to overcome the plant’s immune system and increase the host’s susceptibility. The genetic diversity and composition of T3E repertoires in a large sampling of field strains have yet to be explored on a genomic scale, limiting our understanding of pathogen evolution in an agricultural setting. To this end, we sequenced the genomes of sixty-seven Xanthomonas euvesicatoria (Xe), Xanthomonas perforans (Xp), and Xanthomonas gardneri (Xg) strains isolated from diseased pepper and tomato fields in the southeastern and midwestern United States. T3E repertoires were computationally predicted for each strain and whole genomic phylogenies were employed to understand better the genetic relationship of strains in the collection. From this analysis we detected a division in the Xp population that supported a model whereby a host-range expansion of Xp field strains on pepper is due, in part, to a loss of the T3E AvrBsT. Xp-host compatibility was further studied with the observation that a double deletion of the T3Es AvrBsT and XopQ allowed a gain of host range for Nicotiana benthamiana. Additionally, a single deletion of XopQ expanded the host range of Xe to N. benthamiana, while Xg was a natural pathogen of N. benthamiana. Extensive sampling of field strains and an improved understanding of effector content will aid in efforts to design plant disease resistance strategies targeted against highly conserved effectors.

Xg has emerged recently as the dominant tomato pathogen in parts of the United States and South America. It is responsible for severe crop losses and causes spotting on fruits. Furthermore, Xg appears to be spreading globally. In its repertoire of Type III effectors, Xg possesses a single Transcription Activator Like (TAL) effector protein, AvrHah1, which has previously been shown to confer enhanced water soaked lesions in pepper. TAL effectors act as transcription factors that manipulate expression of target host genes to increase host susceptibility. We investigated the molecular mechanism of AvrHah1-dependent water soaking and the effects of water soaking on enhancing disease severity in tomato. We observed that water from outside the leaf was drawn into the apoplast in Xg-, but not XgΔAvrHah1-, infected tomato, and that water soaking can serve as a mechanism to “ferry” new bacteria into the apoplast. Additionally, AvrHah1 increased the bacterial population present on the surface of diseased tomato leaves. Comparing the transcriptomes of tomato infected with Xg wt vs XgΔAvrHah1 revealed that thousands of genes were differentially upregulated in the presence of AvrHah1. We identified two highly upregulated basic Helix Loop Helix (bHLH) transcription factors with predicted Effector Binding Elements (EBEs) as direct targets of AvrHah1. We mined our RNA-seq data for genes that were highly upregulated but without EBEs and identified two pectin modification genes, a pectate lyase and pectinesterase, which are expressed in response to the bHLH transcription factors and are therefore indirect targets of AvrHah1. Importantly, designer TAL effectors (dTALEs) for the bHLH transcription factors and the pectate lyase complement water soaking in XgΔAvrHah1. By modifying the plant cell wall to enhance water uptake and increase tissue damage, AvrHah1 may improve bacterial dispersal from the apoplast and thereby enhance disease transmission. Understanding lesion development may improve the design of disease tolerance in crops by reducing symptom development and overall pathogen transmission.

Climate change imposes a severe threat to agricultural systems, food security, and human nutrition. Meanwhile, efforts in crop and livestock gene editing have been undertaken to improve performance across a range of traits. Gene editing applications for climate change specifically have converged on four major traits: nutritional quality improvement, yield enhancement, disease tolerance, and abiotic stress tolerance, with the fewest current applications directed towards abiotic stress tolerance. While only few applications of gene editing have been translated to agricultural production thus far, numerous studies in research settings have demonstrated the potential for potent gene editing based solutions to address climate change in the near future. Gene editing of rice (Oryza sativa) specifically holds promise for generating climate resilient foodscapes. Rice is of paramount importance for global nutrition, supplying at least 20% of global calories. However, water scarcity and increased drought severity are anticipated to reduce rice yields globally. Rice stomatal developmental genetics were explored as a mechanism to improve drought resilience while maintaining yield under climate stress. CRISPR/Cas9-mediated knockouts of EPFL10 and STOMAGEN yielded lines with c. 80% and 25% of wild-type stomatal density, respectively. epfl10 lines with moderate reductions in stomatal densities are able to conserve water to similar extents as stomagen lines, but do not suffer from the concomitant reductions in stomatal conductance, carbon assimilation, or thermoregulation observed in stomagen knockouts. Moderate reductions in stomatal densities achieved by editing EPFL10 may be a climate-adaptive approach in rice that can safeguard yield. Editing the paralog of STOMAGEN in other species may provide a means to tune stomatal density in agriculturally important crops beyond rice.

Negative pleiotropic effects of gene editing may be mitigated by editing a single copy of a duplicated gene underlying a trait of interest. However, this approach is limited by a narrow set of duplicated genes whose null phenotype is not deleterious to overall plant fitness. Promoter editing is emerging as an increasingly relevant tool to generate subtle trait variation while mitigating against harmful pleiotropy. We applied a multiplexed, guide design approach informed by bioinformatic analyses to generate genotypic variation in the promoter region of OsSTOMAGEN. Engineered genotypic variation corresponded to continuous variation stomatal density and size. This near-isogenic panel of stomatal variants was leveraged in physiological assays to establish discrete relationships of stomatal density with a range of gas exchange parameters. Developmental plasticity in response to vegetative drought was inhibited in some promoter alleles and in stomagen. Derived stomatal variants can be matched with similarly broad environmental conditions to optimize. Collectively our data suggest a role of promoter editing as a tool for establishing trait variation including phenotypic gain-of-function that can be leveraged for establishing relationships of anatomy and physiology and for crop improvement along diverse environmental clines.

In securing food systems against the severe implications of climate change, gene editing approaches towards the adaptation of rice to abiotic stress has shown promise. An additional approach makes use of gene editing for improving crop quality in crops with existing tolerance. To this end, we sought to improve the safety of the drought-stress-tolerant cassava crop, for human consumption using CRISPR/Cas9. Cassava accumulates cyanogenic glucosides which are human-toxic-metabolites that must be removed to avoid severe human health consequences. Cyanogenic glucosides may play an important physiological role in cassava plants, so eliminating their synthesis entirely may also limit overall productivity. Our work sought to engineer tissue specific accumulation of cyanogenic glucosides by editing, MeCGTR1, a putative systemic transporter using CRISPR/Cas9. cgtr1 lines exhibited depletions of cyanogenic glucosides in upper leaves while maintaining wild-type levels in tuberous roots. Together with a phloem girdling assay, our data indicated a root-to-shoot mode of cyanogenic transport which stands in contrast to previously documented modes of detected movement. Our work provides the first in-vivo validation of a cyanogenic glucoside transporter in cassava providing evidence for the de novo biosynthesis of cyanogenic glucosides in roots.

This Dissertation presents a comprehensive study of how oomycete pathogens are recognized by the plant model organism Arabidopsis thaliana. The key molecular factors in this study include pathogen derived effector molecules, and plant disease resistance genes; specifically, the effector, Arabidopsis thaliana recognized 1 (ATR1) and cognate plant resistance gene, Recognition of Peronospora parasitica 1 (RPP1). Presented data, gathered using genetics, biochemistry, structural biology, natural history and human logic, provide insights into molecular basis for recognition of ATR1 by RPP1. It is demonstrated that the in planta physical association between ATR1 and RPP1 leads to activation of plant immunity. The main regions of RPP1 responsible for effector recognition and downstream activation of immunity are elucidated. Examination of natural variation among ATR1 and RPP1 variants and subsequent heuristic mutational analyses yielded proteins with reversed activity, allowing manipulating the plant to expand its range of pathogen recognition. The 3-D structure of ATR1 was determined and showed that several independent regions could be recognized by RPP1 variants, illustrating adaptive components in plant immunity. Finally, the observed interactions between 83 different Arabidopsis sub-species and 5 strains of its native obligate oomycete pathogen provide show multi-level regulations of host / pathogen interactions, including developmental control of resistance in the host, and the ability of pathogen to suppress recognition of effectors and plant immunity.

Microbial pathogens of plants secrete effector proteins into host cells to suppress basal immunity and manipulate host metabolism, but can be detected by a repertoire of innate immune receptors known as nucleotide-binding leucine rich repeat (NLR) proteins. Though NLRs were identified nearly 25 years ago, are ubiquitous across plant species, and are important components of pest resistance strategies in crops, the molecular mechanisms of NLR function are still unclear. This dissertation presents work on RPP1 (Resistance to Peronospora parasitica), an Arabidopsis NLR receptor that specifically recognizes the effector protein ATR1 from the naturally occurring oomycete pathogen Hyaloperonospora arabidopsidis. Genetic and molecular analysis of ATR1-RPP1 direct association demonstrates a variety of allele-specific recognition surfaces on the tandem WY-domain structure of ATR1. Mutations on these surfaces specifically activate a series of closely related RPP1 homologs, which show surprising structural variation in their recognition domain, a series of C-terminal leucine-rich repeats. Specificity is dictated by LRR binding affinity and full-length receptor sensitivity to activation, as demonstrated by chimeric NLR constructs and specific LRR mutations along a predicted ligand binding surface. Finally, recognized and unrecognized ATR1 alleles are used to probe inter-domain interactions and receptor oligomerization events upon effector binding. In addition, components of downstream signaling are studied using the autoactive, effector-independent TIR signaling domain of RPP1. Mechanistic details for how NLR receptors recognize pathogenic signatures may allow engineering of expanded NLR recognition specificity, yielding broad-spectrum, durable disease resistance in crop plants.

Diseases caused by the family Potyviridae require the interaction of viral genome-linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Disruption of these host genes responsible for Potyviridae susceptibility confers disease resistance. CRISPR/Cas9 has emerged as a robust and specific gene editing technology. As a proof of concept for CRISPR-mediated susceptibility gene targeting, Arabidopsis thaliana was chosen as a model plant system for editing resistance to Turnip mosaic potyvirus (TuMV). CRISPR/Cas9 was employed to generate eif(iso)4e mutants in A. thaliana. eif(iso)4e mutants exhibited resistance to TuMV systemic spread and symptom development. Precise gene editing of A. thaliana eIF(iso)4E was also achieved in somatic cells via the CRISPR/Cpf1 tool.

Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cassava encodes five eIF4E isoforms: eIF4E, eIF(iso)4E-1, eIF(iso)4E-2, novel cap-binding protein-1 (nCBP-1), and nCBP-2. Yeast two-hybrid analysis detected interactions between both CBSV and UCBSV VPg proteins and cassava nCBP-1 and nCBP-2. CRISPR/Cas9-mediated genome editing was employed to generate eif4e, ncbp-1, ncbp-2, and ncbp-1/ncbp-2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp-1/ncbp-2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titer in storage roots relative to wild-type controls. However, full resistance to CBSD was not achieved, suggesting that remaining functional eIF4E isoforms may be compensating for the targeted mutagenesis of nCBP-1 and nCBP-2.

Furthermore, viruses of the family Geminiviridae threaten to global food security through severe crop loss. Tomato yellow leaf curl virus species (TYLCV; genus Begomovirus) cause widespread destruction of the tomato crop. The high levels of genetic variability and persistent insect vector distribution pose significant challenges for conventional and genetic control strategies. In addition to precise genome editing, CRISPR/Cas9 may be employed as a form of molecular immunity against plant DNA viruses. In this study, sgRNA were designed to target a broad range of the TYLCV species, mutagenize a stable region of the TYLCV genome, and disrupt expression of the virus replication machinery. Functionality of multiple TYLCV-targeting CRISPR/Cas9 expression systems was demonstrated in Nicotiana benthamiana. The utility of this TYLCV control strategy was then extended to tomato. CRISPR/Cas9 transgenic tomato lines exhibited reduced TYLCV symptom severity and viral DNA presence. CRISPR-mediated viral interference and viral susceptibility gene mutagenesis reflect the vast potential of the CRISPR technology as a solution to global food challenges.