<b>Tomato receptor like-cytoplasmic kinases regulate plant </b><b>response to pathogens </b>
Creators: name: Hailemariam, Sara Gebremeske
Publisher: name: Purdue University Graduate School
Year: 2024
Subjects: classificationCode: 310805
subject: Plant pathology
classificationCode: 300103
subject: Agricultural molecular engineering of nucleic acids and proteins

description: Plant immunity to pathogens involves a network of genetic, molecular, and cellular processes that culminate in activation of responses that restrict pathogen ingress and reduce diseases symptoms. Mechanisms of pathogen recognition, signaling, and activation of immune responses are well understood especially for (hemi) biotrophic pathogens. By contrast, there is paucity of knowledge on immune signaling for responses to broad host necrotrophic fungi such as <i>Botrytis cinerea (Botrytis</i><i>)</i>. Plant resistance to such pathogens is genetically complex with no complete resistance observed in any host species. However, genetic variation for quantitative resistance has been documented although the molecular mechanisms are poorly understood. In the current study, we focused on functional dissection of components of tomato immune signaling underlying quantitative resistance to <i>Botrytis</i>. Tomato BIK1-family receptor-like cytoplasmic kinases (RLCKs) were studied using gene edited mutants, and subsequent molecular, biochemical, and genomic characterizations of the mutants and the corresponding proteins. In addition, <i>Botrytis</i> induced transcriptome of Arabidopsis and tomato were compared to explain observed differences in their resistance to the fungus. The results from these studies are described in four chapters. Chapter 1 provides a review of RLCKs and their function in plant responses to biotic and abiotic stresses. Chapter 2 presents the genetic, molecular, and biochemical characterization of tomato RLCK, TPK1B RELATED PROTEIN KINASE (TPK09) in fungal resistance and responses to light stress. Chapter 3 highlights observations on the functions of TPK1B RELATED PROTEIN KINASE7 (TPK07) in fungal and bacterial resistance. Lastly, Chapter 4 covers comparative transcriptome analysis of Arabidopsis and tomato responses to <i>Botrytis</i>.Specialized cell-surface receptors mediate the perception of environmental changes. A subset of plant cell surface receptors recognizes pathogen-associated molecular patterns (PAMPs), which are immunogenic pathogen or host-derived molecules, peptides (phytocytokines), lipids, and carbohydrates. PAMPs are perceived by surface receptors designated as pattern recognition receptors (PRRs) that are categorized as receptor like kinases (RLKs) or receptor like proteins (RLPs). Recognition of PAMPs through PRR is an evolutionarily conserved pathway that aids plants in specific recognition of pathogens. The signaling events initiated by PRRs are connected to PRR-associated RLCKs, which amplify the signal and activate other regulatory proteins. PRR-RLCK activation is linked to immune messengers ROS, Ca<sup>+</sup>, and MAPKs based on extensive research in Arabidopsis which also established the paradigm for RLCKs functions in integrating signals from various PRRs. However, insufficient attention was given to RLCK functions in crop plant responses to biotic and abiotic stressors and, thus, limited data exists on RLCKs from crops of agronomic and horticultural significance. To address this gap, we conducted genetic, genomic, and molecular studies on the biological functions of tomato TPK1B RELATED KINASES TPK09 and TPK07.To determine the function of TPK09 and TPK07, mutant alleles of these two RLCKs were generated through CRISPR-Cas9 gene editing. Loss of function mutants of <i>tpk09</i> exhibited increased susceptibility to <i>Botrytis</i> but showed no altered responses to the bacterial pathogen <i>P. syringae</i>. Plants carrying mutant alleles displayed reduced immune gene expression and impaired accumulation of reactive oxygen species in response to chitin and flg22. In addition to <i>Botrytis</i> and several plant hormones, the expression of <i>TPK09</i> gene is induced by light but suppressed by darkness. The exposure of wildtype tomato plants to light-emitting diodes (LEDs) reduced hypocotyl length but <i>tpk09</i> mutants were insensitive. Furthermore, <i>tpk09</i> mutants also exhibited increased accumulation of H<sub>2</sub>O<sub>2</sub> and extensive necrosis, suggesting a disturbance in cellular homeostasis in response to changes in light spectra, ultimately leading to enhanced susceptibility to <i>Botrytis</i>. Although the global impact of TPK09 on <i>Botrytis</i> induced transcriptome was limited, the expression of the tomato negative regulator of cell death SlBI-like1 gene was significantly reduced in the mutant, particularly in response to the combined effect of <i>Botrytis</i> and LED light. The data suggest TPK09 regulates SlBI-like1 gene expression, but the mechanism is unclear. Further, the impairment of the light stress response in <i>tpk09</i> mutants was substantiated by a reduction in chlorophyll content and damage to the photosynthetic machinery, along with a clear reduction in the expression of genes related to light harvesting and photosynthesis. Regulatory network analysis using RNA-seq data identified TPK09 regulated genes related to stress and oxidative damage. This was further supported from proteomic studies of tomato TPK09-HA transgenic plants. Immunoprecipitation coupled with mass spectrometry revealed that proteins associated with photosynthesis and photosystem subunits were enriched in TPK09 overexpressing plant. Our data establishes that TPK09 is required for fungal resistance and light stress by maintaining functional photosynthetic machinery and ROS homeostasis.We also studied the function of tomato TPK07 which is another member of the RLCK-VII family. Through analysis of <i>tpk07</i> mutants, we show that TPK07 it required for resistance to both <i>Botrytis</i> and <i>P. syringae</i> expressing the cysteine protease type III effector AvrPphB which cleaved TPK07 protein. However, further studies are needed to understand the biological function of this cleavage. The expression of TPK07 was induced by <i>Botrytis</i> and the bacterial PAMPs flg22, flg28, and csp22. TPK07 belongs to the RLCK VII-5 subfamily and clusters in the same clade with Arabidopsis PBL34 (AT5G15080), PBL35 (AT3G01300), and PBL36 (AT3G28690) that functions in immunity to <i>P. syringae</i>. In addition, <i>tpk07</i> mutants showed reduced accumulation of ROS in response to chitin and lipopolysaccharide. Moreover, TPK07 is a plasma membrane-localized kinase with a unique N-terminal sequence, offering a research avenue for future structural analysis to understand its contribution in pathogen responses.In the last chapter, we conducted comparative analyses of <i>Botrytis</i> induced transcriptome of Arabidopsis and tomato to explain the observed differences between the two plant species in their resistance to <i>Botrytis</i>. Over the years, we observed tomato is more susceptible to <i>Botrytis</i> than Arabidopsis under the same disease assay conditions, but the mechanism is not known. We sought to gain insight into the immune responses of the two species and identify targets for future functional analyses that can explain the differential pathogen responses. Many separate studies<i> </i>have demonstrated<i> </i>that <i>Botrytis</i> causes extensive transcriptional reprogramming in Arabidopsis and tomato. Our results from the comparative studies of in-house generated data identified differentially expressed genes (DEGs), Gene Ontology terms, and metabolic pathways that are specific or shared between the two species. Interestingly, some genes show distinct expression pattern in tomato and Arabidopsis consistent with previous observation that some genes in the two species show contrasting disease resistance functions. Genes with distinct expression patterns warrant further studies to better understand differences in host immune responses between the two species. In addition, transcription factors (TFs) and regulatory hub genes that could be important for further studies were identified.In sum, our data establishes the functions of two tomato RLCKs in fungal resistance, interactions between fungal resistance and plant responses to light, and the conserved and contrasting gene expression profiles of tomato and Arabidopsis genes, laying the foundation for future studies.