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The maize gene Rp1-D21 is a mutant form of the gene Rp1-D that confers resistance to common rust. Rp1-D21 triggers a spontaneous defense response that occurs in the absence of the pathogen and includes a programed cell death called the hypersensitive response (HR). Eleven plants heterozygous for Rp1-D21, in four different genetic backgrounds, were identified that had chimeric leaves with lesioned sectors showing HR abutting green nonlesioned sectors lacking HR. The Rp1-D21 sequence derived from each of the lesioned portions of leaves was unaltered from the expected sequence whereas the Rp1-D21 sequences from nine of the nonlesioned sectors displayed various mutations, and we were unable to amplify Rp1-D21 from the other two nonlesioned sectors. In every case, the borders between the sectors were sharp, with no transition zone, suggesting that HR and chlorosis associated with Rp1-D21 activity was cell autonomous. Expression of defense response marker genes was assessed in the lesioned and nonlesioned sectors as well as in near-isogenic plants lacking and carrying Rp1-D21. Defense gene expression was somewhat elevated in nonlesioned sectors abutting sectors carrying Rp1-D21 compared with near-isogenic plants lacking Rp1-D21. This suggests that, whereas the HR itself was cell autonomous, other aspects of the defense response initiated by Rp1-D21 were not. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

The maize brachytic2 (br2) gene encodes a protein homologous to the Arabidopsis ABCB1 protein, which actively transports chemically diverse substrates across cellular membranes. This includes indole-3-acetic acid (IAA), the most abundant natural form of auxin, a key regulator of plant growth and tropisms. Defects in auxin transport are responsible for the short stature of br2 mutants. We describe a novel br2 mutant allele present in NC238, an inbred maize of medium stature and demonstrate that the mutant allele br2-NC238 is caused by the insertion of a novel transposon in intron IV of the br2 gene. A tall revertant of NC238 (NC238-rev) appeared spontaneously as a result of excision of the transposon from the br2 gene. This event yielded two completely isogenic versions of the NC238 inbred, one brachytic (NC238-ref) and one tall (NC238-rev). The presence of the transposon in br2-NC238 alters normal splicing of the transcript, producing several splicing forms. One splicing form, ABCB1-T02, was abundantly expressed in NC238-ref, while its expression was very weak in tall NC238-rev plants. Differences between NC238-rev and NC238-ref were also detected in the expression pattern of auxin transporter genes. Morphological changes associated with the br2-NC238 mutant allele included reduced length and increased diameter of the lower internodes. The shortening of plant stature was confirmed in the B73 genetic background by introgression of the br2-NC238. Our results demonstrate that br2-NC238 reduces plant height without altering other morphological traits, and might thus be useful in maize breeding programs when reduction of stature is desired.

The partially dominant, autoactive maize dis- ease resistance gene Rp1-D21 causes hypersensitive response (HR) lesions to form spontaneously on leaves and stems in the absence of pathogen recognition. The maize nested association mapping (NAM) population consists of 25 200-line subpopulations each derived from a cross between the maize line B73 and one of 25 diverse inbred lines. By crossing a line carrying the Rp1-D21 gene with lines from three of these subpopulations and assessing the F1 progeny, we were able to map several novel loci that modify the maize HR, using both single-population quan- titative trait locus (QTL) and joint analysis of all three populations. Joint analysis detected QTL in greater number and with greater confidence and precision than did single population analysis. In particular, QTL were detected in bins 1.02, 4.04, 9.03, and 10.03. We have previously termed this technique, in which a mutant phenotype is used as a ''reporter'' for a trait of interest, Mutant-Assisted Gene Identification and Characterization (MAGIC).

Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance., Author Summary The plant hypersensitive defense response (HR) is a rapid, localized cell death, usually occurring upon the recognition of specific pathogen-encoded molecules and consequent activation of nucleotide binding-leucine rich repeat (NLR) proteins. Rp1-D21, a naturally-occurring mutant caused by the recombination of two NLR genes, confers a ‘lesion mimic’, HR-like phenotype in the absence of pathogen infection and provides a powerful tool to investigate the molecular mechanisms of NLR regulation. Here we report the results of a genetic screen in maize that identified novel mutations abrogating Rp1-D21-induced HR. To characterize the function of Rp1-D21, we transiently expressed Rp1-D21 and various derivatives in Nicotiana benthamiana to observe the resulting levels of HR. We furthermore examined the protein-protein interactions between and within different Rp1-D21 derivatives. We report novel insights into the precise structural requirements for NLR function and determine the function of a previously undefined motif. These insights enable a better understanding of how NLRs regulate the switch between the resting and the active states.