A shortcut in forward genetics: concurrent discovery of mutant phenotype and causal mutation in Arabidopsis M2 families via MAD-mapping

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Forward genetics is a powerful tool to establish phenotype-genotype correlations in virtually all areas of plant biology and has been particularly successful in the model plant Arabidopsis. This approach typically starts with a phenotype in an M2 mutant, followed by identifying a causal DNA change in F2 populations resulting from a cross between the mutant and a wildtype individual. Ultimately, two additional generations are needed to pinpoint causal DNA changes upon mutant identification. We postulated that genome-wide allele frequency distributions within the mutants of M2 families facilitate discrimination of causal versus non-causal mutations, essentially eliminating the need for F2 populations. In a proof-of-principle experiment, we aimed to identify signalling components employed by the executor-type resistance (<jats:italic>R</jats:italic>) protein, Bs4C, from pepper (<jats:italic>Capsicum pubescens</jats:italic>). In a native setting, <jats:italic>Bs4C</jats:italic> is transcriptionally activated by and mediates recognition of the transcription activator-like effector AvrBs4 from the bacterial pathogen <jats:italic>Xanthomonas</jats:italic>. Arabidopsis containing an estradiol-inducible <jats:italic>Bs4C</jats:italic> transgene was used in a conditionally lethal screen to identify second-site suppressor mutations. Whole genome sequencing was used for <jats:underline>M</jats:underline>2 mutant <jats:underline>a</jats:underline>llele-frequency <jats:underline>d</jats:underline>istribution (MAD) mapping in three independent M2 families. MAD-mapping uncovered that all three families harboured mutations in <jats:italic>XRN4</jats:italic>, a novel component of executor R protein pathways. Our work demonstrates that causal mutations observed in forward genetic screens can be identified immediately in M2 families instead of derived F2 families. Notably, the timesaving concept of MAD mapping should be applicable to most crop species and will advance the appeal of forward genetics beyond applications in fundamental research.</jats:p><jats:sec><jats:title>SIGNIFICANCE</jats:title><jats:p>Forward genetics has uncovered numerous genes that govern plant immune reactions. This procedure relies on mutant plants with modified immune reactions followed by identification of causal DNA changes in derived F2 progeny. We developed a novel forward genetics concept where causal DNA changes are identified in the initial M2 mutants, making time consuming establishment of F2 populations obsolete. To confirm the feasibility of the concept, we mutagenized transgenic Arabidopsis seeds containing the cell death executing resistance gene <jats:italic>Bs4C</jats:italic> from pepper. Whole-genome sequencing of identified mutant families that lack a <jats:italic>Bs4C</jats:italic>-dependent cell death revealed the <jats:italic>XRN4</jats:italic> gene as a novel component of <jats:italic>Bs4C</jats:italic>-dependent cell death. This confirms our hypothesis that causal mutations can be identified directly within phenotypically selected mutant families.</jats:p></jats:sec>

The tomato resistance gene Bs4 suppresses leaf watersoaking phenotypes induced by AvrHah1, a transcription activator‐like effector from tomato‐pathogenic xanthomonads

“…Bs3 (Ca02g00940) coding sequence (CDS), triple FLAG‐tag, and T2A self‐cleaving‐peptide (Donnelly et al ., 2001) and an mGFP5 reporter were integrated into estradiol‐inducible promoter (EIP)‐containing pER10 vector (Zuo et al ., 2000) to yield pER10:Bs3‐3xFLAG‐T2A‐mGFP5 construct as described previously (Holmes et al ., 2020). This plasmid was used for stable tomato transformation (Wittmann et al ., 2016).…”