Discovery and validation of host resistance factors for spring black stem and leaf spot disease in Medicago truncatula

Abstract

The model legume, Medicago truncatula, is a self-fertile diploid relative of Medicago sativa (alfalfa). M. truncatula is a valuable resource for understanding legume physiology and plant-microbe interactions. Ascochyta blights cause yield loss in major legume crops. The necrotrophic fungus Ascochyta medicaginicola is the causal agent of spring black stem and leaf spot (SBS) disease, which is one of the most devastating foliar diseases of alfalfa affecting yield, quality, and stand survival in this perennial forage crop. However, host resistance is poorly understood. Quantitative trait loci (QTL) have been described for a variety of agronomic traits to identify targets for plant improvement. In M. truncatula, recessive QTL rnpm1 and rnpm2 were identified in populations generated using M. truncatula genotype HM078 as the resistant parent. In Chapter 1, we generated a genome of HM078 to investigate factors enabling host resistance. We identified 14 candidate genes for disease resistance based on structural variation in QTL regions, with a focus on loss-of-function events in HM078. Next, in Chapter 2 we performed an analysis of the host transcriptome in response to A. medicaginicola infection over time. A total of 192 and 2,908 differentially expressed genes (DEGs) were observed in the resistant (HM078) and susceptible (A17) genotype, respectively. We performed a functional analysis and identified 22 candidate genes for disease resistance based on a variety of factors, such as uniquely upregulated genes in HM078. In Chapter 3, we sought to validate top candidate genes in QTL regions with CRISPR/Cas9-mediated knockouts. We targeted a TIR-NBS-NLR plant disease resistance gene, MtTCAR1, and MtPHO2A in rnpm1, as well as a MtCPR1-like F-box family gene and MtPAM16 in rnpm2. Bi-allelic knockouts of MtTCAR1, MtPHO2A, and MtPAM16 did not affect disease resistance in M. truncatula accession R108. MtCPR1-like mutant plants exhibited a reduction in mean pathogen biomass, increased chlorosis, and variable constitutive expression of pathogenesis-related genes. Interestingly, the constitutive overexpression of an RNA-seq-derived candidate, MtKCS12, enhanced SBS disease resistance significantly, resulting in a ~75% reduction in mean pathogen biomass compared to the null segregant in a detached leaf assay. This study provides insights into omics strategies for investigating disease resistance, as well as gene-editing and transgenic approaches for crop improvement.