The PHD finger protein EDM2 and the two RRM domain proteins EDM3 and IBM2 were shown to preferentially target genes that contain heterochromatin. In my thesis research, I found that only the longer IBM2 isoforms, IBM2L, interacts with both expressed EDM3 isoforms, EDM3S and EDM3L. These interactions positively regulate flowering time and negatively regulate basal immunity. Mutants of EDM3 and IBM2 exhibit a delay of gradually declined expression of the floral suppressor gene FLC during the progression of development. This is negatively correlated with a gradual increase of the transcript levels of the interacting EDM3 and IBM2 isoforms. Moreover, both EDM3L and IBM2L directly target certain defense-associated genes and suppress basal immunity. Lastly, these isoforms are also found to coordinate the floral transition with a gradual repression of basal immunity. Collectively, these findings suggest a functional link between basal immunity and flowering time control. Both processes are clearly coordinated by EDM3L and IBM2L. I further found that only IBM2L can co-localize with both EDM3 isoforms at the COPIA-R7 retrotransposon within the plant immune receptor gene RPP7. Consistently, only IBM2L can rescue RPP7-mediated resistance against Hyaloperonospora arabidopsidis (Hpa) Hiks1 and expression levels of the full-length RPP7-coding transcripts. Like mutants of EDM2, edm3 and ibm2 mutants also exhibit growth defects. We found that EDM2, EDM3 and IBM2 negatively regulate peroxidase activity by downregulating the expression levels of peroxidases. I further found that all three genes promote growth and negatively regulate basal immunity partly by inhibiting peroxidase activity. As growth defects in edm2, edm3 and ibm2 mutants are only partially rescued by suppression of peroxidase activity, there may exist other mechanisms coordinating immunity and growth. By crossing with the sid2-2 and pad4 mutants, which are deficient in SA-mediated immunity, and the transgenic NahG line, which cannot accumulate SA, growth defects in all three mutants are fully or partly rescued. Lastly, we also found that growth defects and enhanced basal immunity can be partly rescued by the longer IBM1 isoform, IBM1L. Collectively, my results show that EDM2, EDM3 and IBM2 have complex roles in coordinating immunity and developmental processes in Arabidopsis.

The generalist soil-borne fungal pathogen Macrophomina phaseolina is known to cause a disease most widely known as charcoal rot. Despite the widespread damage this pathogen has caused on over 500 species of plants including important crops, the mechanism of its virulence is still unclear, and effective methods of control are still lacking. In this study, M. phaseolina interaction with the model plant Arabidopsis thaliana is described using a plate-based infection assay which allows for image analysis-based quantitative assessment of disease progression. From a panel of A. thaliana inbred lines derived from natural populations, we observed significant quantitative deviation in growth inhibition caused by M. phaseolina. In order to gain insight to the genetic architecture behind M. phaseolina resistance, we sought to utilize the natural genetic variation represented in the panel of 87 natural A. thaliana accessions. Genome-wide association approaches combined with RNA-sequencing identified multiple genomic loci and candidate genes which could potentially contribute to the phenotypic variance. Seedling root transcriptomes captured at three time points post-infection revealed that resistant and susceptible A. thaliana genotypes have distinct patterns in defense gene expression. Surprisingly, in the two resistant accessions examined, a rapid decline in up-regulation of many defense genes were observed between 12 and 48 hours post-infection. Genes previously associated with plant immunity, as well as genes involved in processes that have not been seen in M. phaseolina response, were identified in this study, providing a robust pool of candidate genes which will be a useful resource for increasing M. phaseolina tolerance in host plants.

Providing food for human beings is one of the major challenges for the future. Plant diseases cause massive losses in crop production all over the world. Pesticides have been used as a major strategy of crop disease control, however conventional pesticides typically rely on toxic activity leading to environmental problems. Synthetic elicitors can protect plants from diseases by activating host immune responses. The identification and characterization of synthetic elicitors can result in valuable tools for the dissection of the plant defense network as well as leads for the development of environmentally-safe pesticide alternatives.

Synthetic elicitors have been classified and the vast majority of known them belong to the large group of functional SA analogs. Additionally imprimatins, sulfonamides, adipic acid derivatives and jasmonic acid analogs were found to have synthetic elicitor activity.

By high-throughput screening we identified over 100 synthetic elicitors. By using model pathosystems Arabidopsis/Hpa, I functionally characterized two novel synthetic elicitors, BHTC and DPMP and their effects on plant defense pathways. BHTC can induce disease resistance quickly and transiently, has a distinct mode-of- action from already characterized synthetic elicitors. BHTC can enhance root elongation on Arabidopsis when applied at low doses, while induces defense reactions at high doses. This phenomenon is known as hormesis. Transcriptional patterns associated with BHTC-mediated hormesis were different from those associated BHTC-mediated defense. Furthermore, the WRKY70 transcription factor is required for both BHTC-mediated immunity and hormetic root elongation and links plant defense signaling to hormetic developmental responses.

DPMP is one of the strongest synthetic elicitors that were identified by our screening. It also exhibited hormesis effect at low doses and induces disease resistance at high doses. Its activity is fully dependent on NPR1 and partially dependent on WRKY70. Interestingly, two separate moieties of DPMP can independently induce immune responses. While their direct targets in plant defense are still yet to be defined, it is clear that they are powerful tools to dissect plant defense networks as well as develop novel pesticide alternatives

ABSTRACT OF THE DISSERTATION

The Analysis of WRKY70/ LURP1 Dependent Defense Mechanism in Arabidopsis and Tomato

by

Ayesha Baig

Doctor of Philosophy, Graduate Program in Plant Biology

University of California, Riverside, March 2013

Dr. Thomas Eulgem, Chairperson

The Arabidopsis LURP1 gene of LURP gene cluster (Late Up-regulation in Response to Hpa) is a member of a 15-member gene family termed LOR (LURP-one related). Of all Arabidopsis LOR family members, only LURP1 shows an unusually pronounced up-regulation in response to Hyaloperonospora arabidopsidis (Hpa) where as LOR1 shows strong constitutive expression based on microarray data. Reverse genetic studies using transposon insertion mutants revealed an important role of LURP1 in disease resistance mediated by the R-gene RPP5, while LOR1 plays a significant role in basal defense against Hpa. The promoter swap expression lines -1004pLURR1::LOR1 and -1093pLOR1:: LURP1 were constructed to determine if the promoter or slight differences in their protein sequences are important for the differences in their defense-related roles. Confocal microscopy with stably expressed GFP fusion proteins showed GFP-LURP1 and GFP-LOR1 localized at the plasma membrane and in to the nucleus. Homozygous GFP-LURP1 and GFP-LOR1 restore wild type immunity in the respective mutant backgrounds during compatible and incompatible interaction with Hpa. Yeast two hybrid (Y2H) screens with LURP1 and LOR1 as baits identified no significant interacting partners from a screen of 2.3 x 105 cDNA clones.

In Arabidopsis LURP expression is controlled by the WRKY70 transcription factor. Multiple transgenic tomato lines (cultivar VFNT Cherry tomato) containing a Hpa-responsive GUS reporter gene containing a promoter fragment of the LURP member CaBP22 (pCaBP22-333::GUS) , responded to the defense inducing chemicals SA, BTH and DCA indicating that the mechanism of LURP regulation is likely conserved between Arabidopsis and tomato. Phylogenetic analysis revealed that WRKY70, which is a single copy gene in Arabidopsis, has two orthologs in tomato termed SlWRKY70a and SlWRKY70b. I made three silencing constructs to individually or co-silence SlWRKY70a and SlWRKY70b. TRV-VIGS silencing construct in homozygous tomato plants containing pCaBP22-333::GUS reporter gene showed that at least one of the two tomato WRKY70 orthologs is required for mediating responses to BTH or DCA defense induction. Thus, the WRKY70/LURP regulatory module is likely conserved between Arabidopsis and tomato.

Plants are subject to attack by microbial pathogens. This research utilized four distinct plant-pathosystems combined with methods of phytopathology, molecular genetics and chemical biology to study mechanisms of plant immunity against microbial phytopathogens. Chemical biology is the use of small molecule compounds to study biological processes. A previous chemical screen identified 114 compounds, called synthetic elicitors, that activate innate immune responses in the model plant Arabidopsis thaliana (Arabidopsis). Here, experiments conducted with three synthetic elicitors were found to provide disease protection in one or more of the following plant-pathogen interactions: Solanum lycopersicum (tomato) and Pseudomonas syringae pathovar tomato (Pst), Vigna unguiculata (cowpea) and Fusarium oxysporum formae speciales tracheiphilum race 3 (Fot3) or Arabidopsis and Hyaloperonospora arabidopsidis (Hpa). The results of these studies demonstrated the potential power of synthetic elicitors as both tools for molecular/chemical biology research and for direct protection of agricultural crop plants. In addition to synthetic elicitor-applied research, innovative agar plate-based infection assays were developed to study defense responses in Arabidopsis against Macrophomina phaseolina (Mp). Dubbed as a “global destroyer of crops”, the necrotrophic, soil-borne fungus Mp infects more than 500 plant species including many economically important crops. Plant defenses against this powerful pathogen are poorly understood. The novel model phyto-pathosystem was used to quantify Mp biomass growth in roots and to analyze shoot disease severity in different Arabidopsis genotypes, quantitatively assessing host factors affecting the outcome of Mp infections. By comparative profiling of host transcripts in roots of the wild type Arabidopsis accession Col-0, with and without Mp infection, expression changes were uncovered in hundreds of genes potentially contributing to Mp defense mechanisms or putative effector targeting schemes. The results established the Mp-Arabidopsis interaction as a useful model pathosystem, which allowed for application of the vast genomics-related resources of this versatile model plant to the systematic investigation of previously understudied host defenses against a major broad host-spectrum plant pathogen.