DISSECTING THE MOLECULAR BASIS OF BASIL-PERONOSPORA BELBAHRII INTERACTIONS AND GENETIC ENGINEERING FOR DISEASE RESISTANCE

Abstract

Oomycetes form a distinct phylogenetic lineage of eukaryotic microorganisms capable of causing diseases in numerous plants and animals. Downy mildews are among the most devastating phytopathogenic oomycetes that form an obligate biotrophic relationship with their specific host. Peronospora belbahrii causes basil downy mildew (BDM), which is considered one of the most threatening diseases of cultivated basil globally. Sweet basil is the most popular herb used extensively in culinary, cosmetic, and therapeutic industry holding a high economic status. However, its production is in jeopardy due to BDM as disease resistance is lacking in sweet basil varieties and introgression of resistant genes through traditional breeding has faced several challenges. Limited fungicides have shown efficacy on BDM disease with a high risk of evolving fungicide resistant pathogen strains. The obligate biotrophic lifestyle of P. belbahrii and tetraploidy of sweet basil has hindered the study of basil-Peronospora belbahrii molecular-interactions due to lack of effective functional genomic tools. Using two Peronospora belbahrii genes with a putative role in pathogenesis and two sweet basil genes with potential functions in host resistance/susceptibility, studies in this dissertation established a set of functional analysis tools for basil and P. belbahrii studies. With these tools, the roles of the above genes in disease resistance/susceptibility were defined genetically, which offers initial insights into the molecular basis of basil- P. belbahrii interactions. In addition, multiple lines of downy mildew resistant sweet basil plants were generated with potential use in commercial production. Chapter 2 describes the functional characterization of a sweet basil L-type lectin gene, Oblectin 1. Plant lectins are carbohydrate-binding proteins, many of which have been shown to play essential roles in plant immunity. Oblectin 1 expression was found highly induced in a resistant cultivar during infection by P. belbahrii, but not detected in a susceptible one, suggesting its role in resistance to BDM. Transgenic plants ectopically expressing Oblectin 1 in a susceptible cultivar Genoveser were successfully generated through Agrobacterium-mediated transformation with high transformation efficiency. Homozygous transgenic lines conferred partial resistance to P. belbahrii. Through this objective, we developed a highly efficient basil transformation system and genetically defined the role of Oblectin 1 in resistance against P. belbahrii. Chapter 3 demonstrates the applicability of using host-induced gene silencing (HIGS) approach to decipher the role of two candidate pathogenicity-related genes, PbEC1 (Peronospora belbahrii Effector Candidate 1) and PbORCER1 (Peronospora belbahrii Oomycete RxLR-Containing Endoplasmic reticulum Resident 1). HIGS relies on an RNAi-based mechanism by which in planta generated small-interfering RNAs corresponding to a pathogen gene are transmitted to the pathogen during infection via cross-kingdom movement to silence the targeted gene. The candidate genes selected to test the efficacy of HIGS in basil were on the basis of effector features and sequence uniqueness. Transgenic basil plants, expressing inverted repeats of either PbEC1 or PbORCER1 partial coding-sequence flanking a pyruvate orthophosphate dikinase (PDK) intron, were generated. Upon infection, silencing of PbORCER1 was observed in all tested homozygous lines expressing RNAi constructs of PbORCER1, however, silencing of PbEC1 transcripts on homozygous transgenic plants expressing the PbEC1 RNAi construct was not consistently observed likely because its expression was highly induced during infection, which masked the detection of silencing. The pathogen growth on homozygous transgenic plants expressing either RNAi construct was markedly reduced. The results suggest that HIGS is operational during sweet basil-P. belbahrii interactions, and therefore offers a promising tool for functional genomics studies of P. belbahrii and generation of basil downy mildew disease resistance. Chapter 4 illustrates the establishment of an efficient Agrobacterium-delivered CRISPR/Cas9 system for targeted mutagenesis of a basil candidate susceptibility gene, ObDMR1. Two CRISPR/Cas9 constructs were developed, targeting different sites of the ObDMR1 gene. 92% of the transgenic plants displayed successful mutagenesis of ObDMR1 at one target site. ObDMR1 editing in sweet basil was shown to be predominantly heterozygous, but a complete knockout of all alleles was successfully achieved in one line in the first generation of transgenic plants. Transgene-free homozygous plants were obtained in the second generation. Homozygous plants having 1-bp frameshift mutations displayed dwarfism at the early seedling stage but later showed normal growth and development resembling wild type. In addition, knockout of ObDMR1 compromised basil downy mildew susceptibility. Collectively, our data in this chapter demonstrates the success of using CRISPR/Cas9-mediated gene editing to generate complete gene knockout basil mutants, the ease in obtaining transgene-free mutant plants in the successive generation, the development of basil downy mildew resistant plants, and the role of ObDMR1 in basil downy mildew disease development. These results contribute to the acceleration of basil gene functional analysis, molecular breeding, and development of resistant varieties for commercial use.