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Histone methyltransferases regulate responses to biotic and abiotic factors in tomato
thesisposted on 12.10.2021, 14:49 by Carol N BvindiCarol N Bvindi
Plants are constantly exposed to biotic and abiotic factors throughout their developmental stages which threaten their growth and productivity. Environmental stresses limit crop productivity and are likely to increase in severity due to the drastic and rapid changes in global climate. In this project, we studied the genetic factors that contribute to plant adaption to pathogens and other environmental factors in tomato. The results of these are presented in chapters 2-4 of this thesis. Chapter 1 covers background information and the review of the current literature in plant responses to biotic and abiotic stress. Chapter 2 deals with functional analysis of tomato histone methyltransferases SDG33 and SDG34 and their role in plant defense and stress tolerance. Chapter 3 focuses on the role of SDG33 and SDG34 on plant responses to Nitrogen. Finally, Chapter 4 summarizes the results from a reverse genetic screen using CRISPR cas9 genome editing to identify Receptor Like Cytoplasmic Kinases (RLCKs) required for plant resistance to fungal pathogens.
Plant responses to environmental cues are underpinned by rapid and extensive transcriptional reprogramming. Post translational modification of histones orchestrate these reprogramming and cellular responses by altering chromatin structure and establishing permissive or repressive states. Histone lysine methylation (HLM) is a principal modification of chromatin that affects various cellular processes. HLM is mediated by histone methyltransferases (HMTs) that deposit methyl groups to specific lysine residues on n-terminal histones tails. Although it is known that chromatin modifications occur in response to environmental cues, the mechanisms by which this is achieved, and the biological functions of HMTs are poorly understood. The function of tomato histone methyltransferases Set Domain Group (SDG)33 and SDG34 in biotic and abiotic stress responses were studied using tomato mutants generated through CRISPR/cas9 genome editing.
SDG33 and SDG34 genes were induced by pathogens, drought stress, the plant hormones methyl jasmonate, salicylate and abscisic acid. The sdg33 and sdg34 mutants display altered global HLMs. SDG34 is required for global H3K36 and H3K4 mono, di- and tri-methylation while SDG33 is primarily responsible for di- and tri- H3K36 and H3K4 methylation. Tomato SDG33 and SDG34 are orthologues of the Arabidopsis SDG8, an H3K4 and H3K36 methyl transferase previously implicated in plant immunity and plant growth through epigenetic control of Carotenoid Isomerase (CCR2) and other target genes. However, the tomato sdg33 or sdg34 single mutants showed no altered responses to fungal and bacterial pathogens likely due to functional redundancy of the tomato SDG33 and SDG34 genes consistent with their overlapping biochemical activities. Interestingly, tomato SDG33 or SDG34 genes rescued the disease susceptibility and early flowering phenotypes of Arabidopsis sdg8 mutant. Expression of CCR2 gene is completely inhibited in Arabidopsis sdg8 mutant attributed to loss of H3K36 di- and tri methylation at CCR2 chromatin. CCR2 gene expression was partially restored by transgenic expression of tomato SDG33 or SDG34 genes in Arabidopsis sdg8. In tomato, the single CCR2 gene is expressed independent of SDG33 or SDG33 genes suggesting that the genomic targets of the tomato HMTs are different. Unexpectedly, sdg33 and sdg34 plants were more tolerant to osmotic stress, maintain a higher water status during drought which translated to better survival after drought. Tolerance of sdg33 and sdg34 to drought stress is accompanied by higher expression of drought responsive genes. Collectively, our data demonstrate the critical role of tomato HLM in pathogen and stress tolerance likely through the regulation of gene expression.
In parallel, we characterized the role of SDGs in mediating nitrogen responses in tomato. The results are described in Chapter 2. Few studies have focused on the role of histone lysine methylation in regulating changes to nutrient availability. Transcriptome analysis in the shoot and roots showed that SDG33 and SDG34 have both overlapping and distinct regulated targets in tomato. In response to nitrogen, 509 and 245 genes are regulated by both SDG33 and SDG34 in response to nitrogen states in the roots and shoot respectively. In the roots these genes were enriched with GO terms such as ‘regulation of gene expression’, regulation of N metabolism’ and ‘regulation of hormone stimuli’. ‘Response to stimulus’, ‘photosynthesis’ and ‘N assimilation’ were the biological processes significantly enriched in the shoots. Overall, we show that SDG33 and SDG34 are involved in regulating nitrogen responsive gene expression and hence physiological nitrogen responses in the roots and shoots.
We also studied the Set Domain Group 20 (SlSDG20) an orthologue of Arabidopsis SDG25 in tomato. The details of our observations are presented in Chapter 3. SlSDG20 belongs to class III HMTs, it has the SET, Post-SET domain and GYF domain important for proline-rich sequence recognition. SlSDG20 is highly induced by B. cinerea, Methyl Jasmonate and Ethylene. To further understand the functions of SlSDG20 in tomato physiological development and plant immunity we generated slsdg20 knockout mutants through CRSIPR/Cas9. We identified one homozygous slsdg20 mutant with 151bp deletion in an exon immediately before the SET domain. Global methylation assay on the slsdg20 mutant confirmed that SlSDG20 is an H3K4 methyltransferase. The slsdg20 mutant is shorter than the wild type, produce more adventitious shoots causing prolific branching, and produce narrow leaves. Further, the mutant produces abnormal fruit and few seeds that hardly germinate. The slsdg20 mutant is highly susceptible to B. cinerea compared to the wild type. In response to Pst DC3000, slsdg20 mutant plants are comparable of the wild type. Resistance to hrcC strain of Pst DC3000 was impaired in the slsdg20 mutant, suggesting a possible role of SlSDG20 in PTI. In sum, tomato SDG20 is regulates plant immunity and plant growth including fertility.
The final chapter focuses on tomato Receptor like cytoplasmic kinases (RLCKs). Plants perceive the presence of pathogens through Pattern Recognition Receptors (PRR) which are predominantly RLKs, and subsequently recruit RLCKs to signal to downstream regulators of defense responses. Many RLCKs were characterized from Arabidopsis for their role in signalling of responses to bacterial infection. An example of RLCKs is Arabidopsis BIK1 which is implicated in signal transmission of pathogen recognition event at the cell surface. The tomato genome encodes 647 RLK/RLCKs comprising about 2% of its predicted genes. The functions of most of these predicted tomato RLCKs and RLKs have not been determined. Previously, our lab characterized the Arabidopsis BIK1 and tomato TPK1b RLCKs for fungal resistance. Here, we conducted a reverse genetic screen focused on BIK1 and TPK1b related tomato RLCKs to identify a subset with defense functions. Virus induced gene silencing and pathogen assays conducted on 15 RLCKs identified four RLCK genes with potential role in plant immunity. Then, tomato knock out mutants were generated for four RLCK genes through CRISPR/cas9 genome editing to validate the VIGS data. Subsequently, we demonstrated that TPK07, TPK09, TPK011 and TRK04 are required for resistance to B. cinerea. The data are supported by the pathogen induced expression of these genes. Furthermore, trk04 seedlings are impaired in seedling growth responses to Jasmonic acid. Our study establishes that tomato TPK07, TPK09, TPK011 and TRK04 contribute to defense against B. cinerea but their mechanism of function needs to be elucidated in future studies