Exploring wheat-Bipolaris sorokiniana interaction during spot blotch disease

Abstract

Chapter 1: Introduction Wheat is the second most widely grown and consumed food crop of the world after rice, and is the staple food of around 35% of the world’s population. The present wheat production is about 749 million tons (FAO, 2016; http://www.fao.org/faostat) and to feed the world’s ever-growing population with annual growth rate of 2.6%, there will be a requirement to produce about 1040 million tons of wheat in 2020. To reach this target, it is crucial to keep the crop free from various biotic as well as abiotic stresses. In recent years, spot blotch caused by Bipolaris sorokiniana has emerged as a serious threat for wheat cultivation in warmer and humid regions of the world. It causes foliar spot blotch, root rot, black point on grains, head blight and seedling blight of wheat and barley. Estimates of yield losses due to spot blotch are reported to vary from 30-80% and can reach up to 100% under severe infection conditions. In spite of several efforts world over, no wheat variety highly resistant to spot blotch has been released for field cultivation. One of the main reasons for this is that the molecular mechanism behind resistance to spot blotch has not yet been fully understood. In order to develop measures to control plant diseases, it is very important to understand not only the characteristic features of the pathogen, but also the molecular mechanism behind the disease progression. With this purpose, the thesis encompasses the following objectives: Objectives of the study 1. To explore the mechanism of plant-pathogen interaction during spot blotch in susceptible and moderately resistant wheat varieties 2. To understand the mechanism of survival of Bipolaris sorokiniana on exposure to the fungicide propiconazole Chapter 2: Isolation and characterization of Bipolaris sorokiniana isolates from different geographical regions of India B. sorokiniana is a phytopathogenic fungus causing diseases in wheat, barley and other winter cereals. Previous studies involving large numbers of strains collected from around the globe suggest that B. sorokiniana exist as numerous forms of isolates varying in virulence and aggressiveness with specific and nonspecific interactions. B. sorokiniana has high morphological as well as pathological variations. We collected or isolated 12 strains of B. sorokiniana from three different wheat growing geographical regions of India. During microscopic examinations, some cultures were found to be polysporic and hence needed to be purified. Thus, monoconidial cultures were established for seven sporulating isolates of B. sorokiniana. These cultures were characterized at morphological level as well as by sequencing the ITS region of the isolates and confirmed to be B. sorokiniana. Like previous reports, our results also showed high morphological variability among the isolates. However, the morphological variation had no relationship with the geographical background. No correlation was observed between genetic similarity of the isolates and their geographical origin, concluding that the morphological characteristics expression is not conditioned solely by genes. Light microscopy and scanning electron microscopy of the spores showed several variations in conidial size, level of melanization and number of septa. For evaluation of disease reaction, the following reported methods of pathogen inoculation were attempted: leaf painting, sterile seed inoculation and inoculation at Zadok’s scale 12 stage. All these methods had some or other limitations for pathogenicity testing and hence another method, inoculation of germinated seeds, was developed. This method was found to be the best method for high throughput evaluation of pathogenicity as well as screening of germplasms. Our study showed that isolates from the same geographic region and morphological group could show differences in virulence levels.

Chapter 3: Exploring the molecular interaction of wheat-Bipolaris sorokiniana during spot blotch disease Triticum dicoccum (emmer wheat) has superior organoleptic, therapeutic and nutritional qualities. However, dominance by high yielding hexaploid wheat varieties has restricted its cultivation to some niche areas in Europe and other regions including the peninsular India. T. dicoccum is resistant to various biotic stresses and rust diseases but highly susceptible to stripe rust and spot blotch. Spot blotch has become a major constraint in T. dicoccum cultivation in India. The hemibiotrophic disease cycle of this pathogen is observed only in the susceptible host. Interactive transcriptome sequencing is gaining importance in plant pathogen interaction studies and has enabled simultaneous analysis of expression of plant as well as pathogen genes. Similarly, next generation sequencing has enabled genome sequencing of organisms to a great extent. With the availability of the reference genome sequences from plants as well as pathogen, it has become much easier to align the reads from RNA-seq data and hence expression quantification. In order to explore the interaction, we performed global transcriptome analysis of spot blotch susceptible variety, DDK 1025 and a moderate resistant variety, Chirya 3 upon pathogen inoculation using Illumina HiSeq platform. To understand the infection process and mechanism of disease progression, we performed differential gene expression analysis of spot blotch susceptible variety, DDK 1025 upon pathogen inoculation. A time series comparative study was performed to understand the biotrophic (1 dpi, days post inoculation), early necrotrophic (4 dpi) and necrotrophic phase (6 dpi) responses. The numbers of differentially expressed genes (DEGs) from three stages were 1810, 1562 and 2908 individually. GO annotations were obtained using Blast2GO for 75.63%, 70% and 73.89% of these DEGs respectively. GO enrichment was performed using agriGo online tool (http://bioinfo.cau.edu.cn/agriGO/analysis.php) using Triticum aestivum transcript ID v2.2 as the reference. Biological processes associated with carbohydrate metabolic process, response to abiotic stress, photosynthesis, cell death, regulation of gene expression, secondary metabolic process and generation of precursor metabolites were enriched. Under molecular function category carbohydrate binding, catalytic activity, enzyme regulator activity, protein binding and hydrolase activity was enriched. Although cellular component distribution showed all cellular parts including endoplasmic reticulum, plastid etc., extracellular region was profoundly enriched. Since acceptable annotation of T. aestivum genome was not available, insights into functional annotation were achieved using blast against Oryza sativa japonica group using the STRING platform v10.5 (https://string-db.org/). Pfam enrichment was performed to gain comprehensive understanding about the gene families involved in the infection process. Pathways intricate to this interaction mechanism were explored by KEGG enrichment of DEGs using ClueGo (cytoscape plugin) using O. sativa blast hits. After several enrichments and annotations, major components involved in the interaction were recognized as glycolysis, phenylpropanoid biosynthesis, protein processing in endoplasmic reticulum, photosynthesis, glyoxylate and dicarboxylate metabolism, heat shock proteins, protein kinases and defense response genes like chitinases and hydrolases. Down-regulation of several defense responsive genes in biotrophic phase suggests the contribution of effector mediated susceptibility. Glutathione metabolism mediated regulation of glycolysis and pentose phosphate pathway was identified. Differential expressions of multiple components of ubiquitin mediated proteolysis emphasize their role in hormone signal transduction during spot blotch. Of these DEGs, 177 genes were differentially expressed across all the three time points irrespective of the phases. Co-expression analysis using k-means clustering showed six patterns. Annotations showed that these genes had activities like chitin catabolic process, defense response to fungus, Bowman-Birk proteinase inhibitor and phenylalanine ammonia-lyase. Further information about the significance of these genes during interaction with the pathogen needs to be revealed using over/under expression experiments. Likewise, to recognize the resistance phenomenon, we sequenced the transcriptome of spot blotch resistant (T. aestivum) hexaploid variety, Chirya 3 upon B. sorokiniana inoculation. Differential expression analysis was performed for three stages i.e. 1 dpi, 4 dpi and 6 dpi, which depicts the biotrophic, early necrotrophic and necrotrophic phases in the spot blotch susceptible variety. Our results showed that the number of upregulated genes was higher than downregulated genes. GO annotation was obtained for 64.38%, 66.6% and 64.25% genes from the DEGs. A higher number of genes were unannotated and were found to have significantly higher fold change expression. This suggests that these genes with unknown function could be novel defense responsive genes from wheat. Comparison of gene ontology enrichment showed that biological processes like photosynthesis and cell death were affected in susceptible variety but not in the resistant variety. Whereas, enhanced activity of extracellular proteinase inhibitors and peroxidases was observed in the resistant variety. Thus, early recognition and activation of defense pathways in resistant variety appears to hinder pathogen growth, survival and hence infection. The results from transcriptome sequencing analyses demand confirmation using other complementary techniques like the quantitative reverse transcriptase polymerase chain reaction (qRT PCR), which is an indispensable tool for gene expression analyses. The most adopted method for relative quantification of gene expression in qRT PCR is based on the DDCt method. However, accurate quantification by this method requires an appropriate internal reference gene with stable expression across all or most of the experimental tissues. Selecting an appropriate internal reference gene is very important to elucidate the target gene expression reliably. Several housekeeping genes including 18S rRNA, ACTIN, GAPDH and EF-1α have been proposed as standard reference genes for qRT PCR studies. However, in case of plant pathogen interaction analyses, selection of an appropriate reference gene is even more crucial due to the presence of RNA from both the plant as well as the pathogen in the infected tissues. As several of these genes are also present in the fungal pathogen genome, this could result in unintended cross amplification; which can cause improper quantification of the target genes. Hence, we aimed to identify a wheat gene with the most stable expression and unique primers, which would selectively amplify only the wheat gene and not the pathogen gene, providing accurate quantification of the target genes. Hence, we evaluated six previously reported genes with expression stability under different conditions using the wheat-Bipolaris sorokiniana system. We employed various statistical analysis methods, based on which, we identified two most stable genes, ubiquitin conjugation enzyme (ULE) and phytochelatin synthase (PCS) as the best reference genes for qRT-PCR based quantification in wheat pathosystem. We further confirmed the expression of several candidate defense genes in wheat using ULE as the reference gene. However, both the genes can be used either individually or together as internal reference genes.

Chapter 4: Global gene expression analysis of Bipolaris sorokiniana after exposure to propiconazole Integrated disease management has been proposed to control the spot blotch disease. However, due to the unavailability of spot blotch resistant wheat varieties, the application of foliar fungicide is the most widely practiced measure. Propiconazole is a commonly used azole fungicide to manage the spot blotch disease in the field. However, due to its fungistatic mode of action, there is a possibility of emergence of fungicide resistant pathogen strains. Several mechanisms are reported for azole resistance in fungi. However, the strategies vary in different fungi. Resistance to the fungicide could be attributed to multiple molecular components in the fungus. Moreover, azoles have multiple modes of action out of which few are not explored yet. Global transcriptomics analysis of the pathogen after exposure to sub-lethal doses of the fungicide can reveal the mechanism of survival as well as the mode of action of the azoles in the fungi. Hence, a time series gene expression analysis was performed using RNA-seq. Transcriptome analysis using various tools showed overexpression of the target genes in the sterol biosynthesis pathway of the pathogen. In addition, this study also revealed altered expression of several metabolic pathways, transporters and stress regulators in the pathogen. The use of multiple analysis tools for transcriptomics analysis provided additional confidence on the observed results. The observed results were validated using qRT-PCR. We explored three strategies in B. sorokiniana against propiconazole stress: i) overexpression of target enzymes, ii) increased expression of transporter genes, and iii) expression modulation of stress responsive factors. This study revealed several novel putative targets such as ent-kaurene oxidase, ligninase lg6 precursor and spore germination protein. These genes help the fungi to overcome stresses and survive. Hence, the drugs targeting these genes can be developed, which are expected to impair the stress tolerance and hence survival of the pathogen. However, resistance is a polygenic phenomenon and to understand the functional contribution of each gene, knockout/knockdown studies are suggested.

Chapter 5: Conclusions and Future Prospects Spot blotch is an emerging disease causing yield losses of economically important cereals. The worldwide distribution of the causal agent, B. sorokiniana makes it of global concern. In the present study, we aimed to explore the mechanism of interaction of the pathogen with spot blotch susceptible and resistant wheat varieties. Initially, we established monoconidial cultures of seven isolates of B. sorokiniana collected from three different wheat growing regions of India and characterized them at morphological and molecular level. Although pathogenicity cannot directly be correlated with morphology on culture media, melanization level might be considered an important aspect in determining the level of virulence. Effector mediated downregulation of innate immunity and delayed response by plants leads to successful establishment of the pathogen in susceptible variety. We found that differential expression of ubiquitin mediated proteolysis played a key role in development of the disease in susceptible variety. On the contrary, proteinase inhibitors and peroxidase secretion led to effective elimination of pathogen in the resistant variety. Genotypes with higher expression of these genes are likely to provide improved resistance against the spot blotch disease. As integrated disease management is a sustainable approach which also includes judicial use of fungicides to control the disease; we explored novel targets for developing efficient fungicides. However, essentiality of these genes for the pathogen survival needs to be confirmed through knock-out/down studies. Overall, this study helped in understanding the molecular paradigm of spot blotch disease in wheat. The outcome of this study will assist in advancement of controlling measures against spot blotch.