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Bidirectionality effects can be a significant confounding factor when measuring hyperspectral reflectance data. The bidirectional reflectance distribution function (BRDF) can effectively characterize the reflectivity of surfaces to correct remote sensing measurements. However, measuring BRDFs can be time-consuming, especially when collecting Mueller matrix BRDF (mmBRDF) measurements of a surface via conventional goniometric techniques. In this paper, we present a system for collecting mmBRDF measurements using static optical fiber detectors that sample the hemisphere surrounding an object. The entrance to each fiber contains a polarization state analyzer configuration, allowing for the simultaneous acquisition of the Stokes vector intensity components at many altitudinal and azimuthal viewing positions. We describe the setup, calibration, and data processing used for this system and present its performance as applied to mmBRDF measurements of a ground glass diffuser.

The pattern-triggered immune (PTI) response in plants is caused by the recognition of conserved microbe‐ or pathogen‐associated molecular patterns (MAMPs) by plant pattern recognition receptors at the cell surface. The goal of this study was to develop a simple, robust assay to quantify the PTI response in maize and to determine whether it could be used to predict levels of disease resistance. Flg22, an epitope derived from bacterial flagellin, is a commonly studied MAMP. We developed a seedling growth retardation (SGR) assay by which we could measure growth retardation in maize seedlings exposed to the bacterial MAMP flg22. We observed variation across 21 maize inbred lines. We used 161 lines from a recombinant inbred line (RIL) population derived from a cross between the lines CML228 (a high responder) and B73 (a low responder) to map quantitative trait loci (QTL) for this response. We found heritable variation in the RIL population and identified flg22 response QTL on chromosomes 1, 2, and 8. We did not observe strong correlations between SGR traits and levels of flg22-induced reactive oxygen production or with other disease resistance or defense response traits we had previously measured in the same population. We discuss the implications of these findings. [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 .

Plant disease resistance proteins (R‐proteins) detect specific pathogen‐derived molecules, triggering a defence response often including a rapid localized cell death at the point of pathogen penetration called the hypersensitive response (HR). The maize Rp1‐D21 gene encodes a protein that triggers a spontaneous HR causing spots on leaves in the absence of any pathogen. Previously, we used fine mapping and functional analysis in a Nicotiana benthamiana transient expression system to identify and characterize a number of genes associated with variation in Rp1‐D21‐induced HR. Here we describe a system for characterizing genes mediating HR, using virus‐induced gene silencing (VIGS) in a maize line carrying Rp1‐D21. We assess the roles of 12 candidate genes. Three of these genes, SGT1, RAR1, and HSP90, are required for HR induced by a number of R‐proteins across several plant–pathogen systems. We confirmed that maize HSP90 was required for full Rp1‐D21‐induced HR. However, suppression of SGT1 expression unexpectedly increased the severity of Rp1‐D21‐induced HR while suppression of RAR1 expression had no measurable effect. We confirmed the effects on HR of two genes we had previously validated in the N. benthamiana system, hydroxycinnamoyltransferase and caffeoyl CoA O‐methyltransferase. We further showed the suppression the expression of two previously uncharacterized, candidate genes, IQ calmodulin binding protein (IQM3) and vacuolar protein sorting protein 37, suppressed Rp1‐D21‐induced HR. This approach is an efficient way to characterize the roles of genes modulating the hypersensitive defence response and other dominant lesion phenotypes in maize., In this study we used a virus‐induced gene silencing technique to characterize the roles of 12 genes in controlling the hypersensitive cell death defence response in maize.

Background The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9). Results In this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression. Conclusions We anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant.

Macroorganisms' genotypes shape their phenotypes, which in turn shape the habitat available to potential microbial symbionts. This influence of host genotype on microbiome composition has been demonstrated in many systems; however, most previous studies have either compared unrelated genotypes or delved into molecular mechanisms. As a result, it is currently unclear whether the heritability of host-associated microbiomes follows similar patterns to the heritability of other complex traits. We take a new approach to this question by comparing the microbiomes of diverse maize inbred lines and their F1 hybrid offspring, which we quantified in both rhizosphere and leaves of field-grown plants using 16S-v4 and ITS1 amplicon sequencing. We show that inbred lines and hybrids differ consistently in the composition of bacterial and fungal rhizosphere communities, as well as leaf-associated fungal communities. A wide range of microbiome features display heterosis within individual crosses, consistent with patterns for nonmicrobial maize phenotypes. For leaf microbiomes, these results were supported by the observation that broad-sense heritability in hybrids was substantially higher than narrow-sense heritability. Our results support our hypothesis that at least some heterotic host traits affect microbiome composition in maize.

Calcium (Ca) is an essential plant nutrient, required for signaling, cell wall fortification and growth and development. Calcium deficiency (Ca-deficiency) in maize causes leaf tip rot and a so-called “bull-whipping” or “buggy-whipping” phenotype. Seedlings of the maize line B73 displayed these Ca-deficiency-like symptoms when grown in the greenhouse with excess fertilizer during the winter months, while seedlings of the Mo17 maize line did not display these symptoms under the same conditions. These differential phenotypes could be recapitulated in ‘mini-hydroponic’ systems in the laboratory in which high ammonium, but not nitrate, levels induced the symptoms in B73 but not Mo17 seedlings. Consistent with this phenotype being caused by Ca-deficiency, addition of Ca2+ completely relieved the symptoms. These data suggest that ammonium reduces the seedling’s ability to absorb calcium, which causes the Ca-deficiency phenotype, and that this trait varies among genotypes. A recombinant inbred line (RIL) population derived from a B73 x Mo17 cross was used to map quantitative trait loci (QTL) associated with the Ca-deficiency phenotype. QTL associated with variation in susceptibility to Ca-deficiency were detected on chromosomes 1, 2, 3, 6 which explained between 3.30–9.94% of the observed variation. Several genes predicted to bind or be activated by calcium map to these QTL on chromosome 1, 2, 6. These results describe for the first time the genetics of Ca-deficiency symptoms in maize and in plants in general.

Herbaspirillum rubrisubalbicans is the causal agent of red stripe disease (RSD) and mottle stripe disease of sorghum and sugarcane, respectively. In all, 63 genotypes of Sorghum bicolor were inoculated with H. rubrisubalbicans, with 59 showing RSD symptoms. Quantitative trait loci (QTL) analysis in a recombinant inbred line (RIL) population identified several QTL associated with variation in resistance to RSD. RNA sequencing analysis identified a number of genes whose transcript levels were differentially regulated during H. rubrisubalbicans infection. Among those genes that responded to H. rubrisubalbicans inoculation were many involved in plant–pathogen interactions such as leucine-rich repeat receptors, mitogen-activated protein kinase 1, calcium-binding proteins, transcriptional factors (ethylene-responsive element binding factor), and callose synthase. Pretreatment of sorghum leaves with the pathogen-associated molecular pattern (PAMP) molecules flg22 and chitooctaose provided protection against subsequent challenge with the pathogen, suggesting that PAMP-triggered immunity plays an important role in the sorghum immunity response. These data present baseline information for the use of the genetically tractable H. rubrisubalbicans–sorghum pathosystem for the study of innate immunity and disease resistance in this important grain and bioenergy crop. Information gained from the use of this system is likely to be informative for other monocots, including those more intractable for experimental study (e.g., sugarcane).

Maize is one of the major crops in the world; however, diseases caused by various pathogens seriously affect its yield and quality. The maize Rp1-D21 mutant (mt) caused by the intragenic recombination between two nucleotide-binding, leucine-rich repeat (NLR) proteins, exhibits autoactive hypersensitive response (HR). In this study, we integrated transcriptomic and metabolomic analyses to identify differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) in Rp1-D21 mt compared to the wild type (WT). Genes involved in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) were enriched among the DEGs. The salicylic acid (SA) pathway and the phenylpropanoid biosynthesis pathway were induced at both the transcriptional and metabolic levels. The DAMs identified included lipids, flavones, and phenolic acids, including 2,5-DHBA O-hexoside, the production of which is catalyzed by uridinediphosphate (UDP)-dependent glycosyltransferase (UGT). Four maize UGTs (ZmUGTs) homologous genes were among the DEGs. Functional analysis by transient co-expression in Nicotiana benthamiana showed that ZmUGT9250 and ZmUGT5174, but not ZmUGT9256 and ZmUGT8707, partially suppressed the HR triggered by Rp1-D21 or its N-terminal coiled-coil signaling domain (CCD21). None of the four ZmUGTs interacted physically with CCD21 in yeast two-hybrid or co-immunoprecipitation assays. We discuss the possibility that ZmUGTs might be involved in defense response by regulating SA homeostasis.

Many correlations exist between spectral reflectance and various phenotypic responses from plants. Of interest to us are structural characteristics; namely, how the various spectral and polarimetric components may correlate to underlying environmental, metabolic, and genotypic differences among plant varieties within a given species. In this paper, we overview a portable Mueller matrix imaging spectropolarimeter that has been optimized for field use. Key aspects to the design included minimizing the measurement time while maximizing signal-to-noise ratio with low systematic errors. These goals must be achieved while maintaining an imaging capability across multiple measurement wavelengths, spanning the blue to near-infrared spectral region. To this end, we will review our optimization procedure, simulations, and experimental results, including preliminary field data taken from our summer 2021 field trials.

Hybrids account for nearly all commercially planted varieties of maize and many other crop plants because crosses between inbred lines of these species produce first-generation [F(1)] offspring that greatly outperform their parents. The mechanisms underlying this phenomenon, called heterosis or hybrid vigor, are not well understood despite over a century of intensive research. The leading hypotheses—which focus on quantitative genetic mechanisms (dominance, overdominance, and epistasis) and molecular mechanisms (gene dosage and transcriptional regulation)—have been able to explain some but not all of the observed patterns of heterosis. Abiotic stressors are known to impact the expression of heterosis; however, the potential role of microbes in heterosis has largely been ignored. Here, we show that heterosis of root biomass and other traits in maize is strongly dependent on the belowground microbial environment. We found that, in some cases, inbred lines perform as well by these criteria as their F(1) offspring under sterile conditions but that heterosis can be restored by inoculation with a simple community of seven bacterial strains. We observed the same pattern for seedlings inoculated with autoclaved versus live soil slurries in a growth chamber and for plants grown in steamed or fumigated versus untreated soil in the field. In a different field site, however, soil steaming increased rather than decreased heterosis, indicating that the direction of the effect depends on community composition, environment, or both. Together, our results demonstrate an ecological phenomenon whereby soil microbes differentially impact the early growth of inbred and hybrid maize.

Target leaf spot (TLS) of sorghum, a foliar disease caused by the necrotrophic fungus Bipolaris cookei (also known as Bipolaris sorghicola), can affect grain yield in sorghum by causing premature drying of leaves and defoliation. Two sorghum recombinant inbred line (RIL) populations, BTx623/BTx642 and BTx623/SC155-14E, were assessed for TLS resistance in replicated trials. Using least square mean trait data, four TLS resistance QTL were identified, two in each population. Of these, three were previously unidentified while a major QTL on chromosome 5 in the BTx623/BTx642 RIL population corresponded to the previously identified TLS resistance gene ds1. A set of sorghum lines were assessed for production of reactive oxygen species induced by treatment with the microbe-associated molecular pattern (MAMP) flg22 (a derivative of flagellin). Flg22-induced ROS production varied between lines in a consistent fashion. One QTL associated with variation in the flg22 response was detected in the RIL populations. No evidence was found to link variation in the MAMP response to variation in TLS resistance

Southern Leaf Blight (SLB), Northern Leaf Blight (NLB), and Gray Leaf Spot (GLS) caused by Cochliobolus heterostrophus, Setosphaeria turcica, and Cercospora zeae-maydis respectively, are among the most important diseases of corn worldwide. Previously, moderately high and significantly positive genetic correlations between resistance levels to each of these diseases were identified in a panel of 253 diverse maize inbred lines. The goal of this study was to identify loci underlying disease resistance in some of the most multiple disease resistant (MDR) lines by the creation of chromosome segment substitution line (CSSL) populations in multiple disease susceptible (MDS) backgrounds. Four MDR lines (NC304, NC344, Ki3, NC262) were used as donor parents and two MDS lines (Oh7B, H100) were used as recurrent parents to produce eight BC3F4:5 CSSL populations comprising 1,611 lines in total. Each population was genotyped and assessed for each disease in replicated trials in two environments. Moderate to high heritabilities on an entry mean basis were observed (0.32 to 0.83). Several lines in each population were significantly more resistant than the MDS parental lines for each disease. Multiple quantitative trait loci (QTL) for disease resistance were detected for each disease in most of the populations. Seventeen QTL were associated with variation in resistance to more than one disease (SLB/NLB: 2; SLB/GLS: 7; NLB/GLS: 2 and 6 to all three diseases). For most populations and most disease combinations, significant correlations were observed between disease scores and also between marker effects for each disease. The number of lines that were resistant to more than one disease was significantly higher than would be expected by chance. Using the results from individual QTL analyses, a composite statistic based on Mahalanobis distance (Md) was used to identify joint marker associations with multiple diseases. Across all populations and diseases, 246 markers had significant Md values. However further analysis revealed that most of these associations were due to strong QTL effects on a single disease. Together, these findings reinforce our previous conclusions that loci associated with resistance to different diseases are clustered in the genome more often than would be expected by chance. Nevertheless true MDR loci which have significant effects on more than one disease are still much rarer than loci with single disease effects. © 2019 by the Genetics Society of America. Funding for the work was provided by USDA-ARS, the Corn Growers’ Association of North Carolina and by NSF grant #1127076 to RJW and PBK. LLZ was supported by a grant from Fulbright Colombia and COLCIENCIAS. We thank Cathy Herring and the staff at Central crops for excellent field support, Shannon Sermons, David Rhyne and Greg Marshall for their technical support. We thank Daniel Gorman, David Bubeck and DuPont-Pioneer for their assistance with field trials in Andrews NC. We thank Jim Holland and Marc Cubeta for their advice and for reviewing the manuscript.

The maize protein Rp1-D21, an autoactive derivative of the Rp1-D nucleotide-binding leucine rich repeat (NLR) disease resistance protein, triggers a spontaneous hypersensitive cell death defense response (HR). ZmCER9, a member of a class of E3 ligases involved in the endoplasmic reticulum associated degradation (ERAD) protein quality control system, is a suppressor of the Rp21-D21 HR phenotype. ZmCER9, an active E3 ligase, localizes to the ER, consistent with a role in ERAD. It physically interacts with and mediates the proteasome-dependent degradation of both Rp1-D21 and another autoactive Rp1-D derivate but neither interacts with nor affects the protein levels of three non-autoactive Rp1-D derivatives. ZmCER9 also suppresses the activity and directs the degradation of autoactive NLRs from Arabidopsis and barley. We describe a novel, and possibly general, mechanism that specifically degrades activated NLRs to suppress the defense response after activation.

The plant hypersensitive response (HR), a rapid cell death at the point of pathogenesis, is mediated by nucleotide‐binding site, leucine‐rich repeat (NLR) resistance proteins (R‐proteins) that recognize the presence of specific pathogen‐derived proteins. Rp1‐D21 is an autoactive maize NLR R‐protein that triggers HR spontaneously. We previously mapped loci associated with variation in the strength of HR induced by Rp1‐D21. Here we identify the E3 ligase ZmMIEL1 as the causal gene at a chromosome 10 modifier locus. Transient ZmMIEL1 expression in Nicotiana benthamiana reduced HR induced by Rp1‐D21, while suppression of ZmMIEL1 expression in maize carrying Rp1‐D21 increased HR. ZmMIEL1 also suppressed HR induced by another autoactive NLR, the Arabidopsis R‐protein RPM1D505V, in N. benthamiana. We demonstrated that ZmMIEL1 is a functional E3 ligase and that the effect of ZmMIEL1 was dependent on the proteasome but also that levels of Rp1‐D21 and RPM1D505V were not reduced when coexpressed with ZmMIEL1 in the N. benthamiana system. By comparison to a similar system in Arabidopsis, we identify ZmMYB83 as a potential target of ZmMIEL1. Suppression of ZmMYB83 expression in maize lines carrying Rp1‐D21 suppressed HR. Suppression of ZmMIEL1 expression caused an increase in ZmMYB83 transcript and protein levels in N. benthamiana and maize. Using coimmunoprecipitation and bimolecular fluorescence complementation assays, we demonstrated that ZmMIEL1 and ZmMYB83 physically interacted. Additionally, ZmMYB83 and ZmMIEL1 regulated the expression of a set of maize very long chain fatty acid (VLCFA) biosynthetic genes that may be involved in regulating HR., We present evidence that induction of the hypersensitive defence response by the maize Rp1‐D21 resistance protein requires the ZmMYB83 transcription factor, which is targeted for degradation by the ZmMIEL1 E3 ligase protein.

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 past few decades have seen major discoveries in the field of molecular plant-microbe interactions. As the result of technological and intellectual advances, we are now able to answer questions at a level of mechanistic detail that we could not have imagined possible 20 years ago. The MPMI Editorial Board felt it was time to take stock and reassess. What big questions remain unanswered? We knew that to identify the fundamental, overarching questions that drive our research, we needed to do this as a community. To reach a diverse audience of people with different backgrounds and perspectives, working in different areas of plant-microbe interactions, we queried the more than 1,400 participants at the 2019 International Congress on Molecular Plant-Microbe Interactions meeting in Glasgow. This group effort resulted in a list of ten, broad-reaching, fundamental questions that influence and inform our research. Here, we introduce these Top 10 unanswered questions, giving context and a brief description of the issues. Each of these questions will be the subject of a detailed review in the coming months. We hope that this process of reflecting on what is known and unknown and identifying the themes that underlie our research will provide a framework to use going forward, giving newcomers a sense of the mystery of the big questions and inspiring new avenues and novel insights. Fil: Harris, Jeanne M.. University of Vermont; Estados Unidos Fil: Balint Kurti, Peter. Nc State University; Estados Unidos Fil: Bede, Jacqueline C.. McGill University; Canadá Fil: Day, Brad. Michigan State University; Estados Unidos Fil: Gold, Scott. University of Georgia; Estados Unidos Fil: Goss, Erica M.. University of Florida; Estados Unidos Fil: Grenville Briggs, Laura J.. Swedish University of Agricultural Sciences; Suecia Fil: Jones, Kathryn M.. Florida State University; Estados Unidos Fil: Wang, Aiming. Agriculture and Agri-Food Canada; Canadá Fil: Wang, Yuanchao. Nanjing Agricultural University; China Fil: Mitra, Raka M.. Carleton College; Estados Unidos Fil: Sohn, Kee Hoon. Pohang University of Science and Technology; Corea del Sur Fil: Alvarez, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina

Hybrids account for nearly all commercially planted varieties of maize and many other crop plants, because crosses between inbred lines of these species produce F1 offspring that greatly outperform their parents. The mechanisms underlying this phenomenon, calledheterosisor hybrid vigor, are not well understood despite over a century of intensive research (1). The leading hypotheses—which focus on quantitative genetic mechanisms (dominance, overdominance, and epistasis) and molecular mechanisms (gene dosage and transcriptional regulation)—have been able to explain some but not all of the observed patterns of heterosis (2, 3). However, possible ecological drivers of heterosis have largely been ignored. Here we show that heterosis of root biomass and germination in maize is strongly dependent on the belowground microbial environment. We found that, in some cases, inbred lines perform as well by these criteria as their F1 offspring under sterile conditions, but that heterosis can be restored by inoculation with a simple community of seven bacterial strains. We observed the same pattern for seedlings inoculated with autoclavedvs.live soil slurries in a growth chamber, and for plants grown in fumigatedvs.untreated soil in the field. Together, our results demonstrate a novel, ecological mechanism for heterosis whereby soil microbes generally impair the germination and early growth of inbred but not hybrid maize.

Similar progressive leaf lesion phenotypes, named conring for "concentric ring", were identified in 10 independently-derived maize lines. Complementation and mapping experiments indicated that the phenotype had the same genetic basis in each line - a single recessive gene located in a 1.1 Mb region on chromosome 2. Among the 15 predicted genes in this interval, Zm00001d003866 (subsequently renamed Conring or Cnr) had insertions of four related 138 bp transposable element (TE) sequences at precisely the same site in exon 4 in nine of the 10 cnr alleles. The tenth cnr allele had a distinct insertion of 226 bp of in exon 3. Genetic evidence suggested that the 10 cnr alleles were independently-derived, and arose during the derivation of each line. The four TEs, named COINa (for COnring INsertion) through COINd, have not been previously characterized and consist entirely of imperfect 69 bp terminal inverted repeats (TIRs) characteristic of the Foldback class of TEs. They belong to three clades of a family of maize TEs comprising hundreds of sequences in the genome of the B73 maize line. COIN elements preferentially insert at TNA sequences with a preference for C and G nucleotides in the immediately flanking 5' and 3' regions, respectively. They produce a three base target site duplication and do not have homology to other characterized TEs. We propose that Cnr is an unstable gene that is insertionally mutated at high frequency, most commonly due to COIN element insertions at a specific site in the gene.

Plants usually employ resistance (R) genes to defend against the infection of pathogens, and most R genes encode intracellular nucleotide-binding, leucine-rich repeat (NLR) proteins. The recognition between R proteins and their cognate pathogens often triggers a rapid localized cell death at the pathogen infection sites, termed the hypersensitive response (HR). Metacaspases (MCs) belong to a cysteine protease family, structurally related to metazoan caspases. MCs play crucial roles in plant immunity. However, the underlying molecular mechanism and the link between MCs and NLR-mediated HR are not clear. In this study, we systematically investigated the MC gene family in maize and identified 11 ZmMCs belonging to two types. Further functional analysis showed that the type I ZmMC1 and ZmMC2, but not the type II ZmMC9, suppress the HR-inducing activity of the autoactive NLR protein Rp1-D21 and of its N-terminal coiled-coil (CCD21 ) signaling domain when transiently expressed in Nicotiana benthamiana. ZmMC1 and ZmMC2 physically associate with CCD21 in vivo. We further showed that ZmMC1 and ZmMC2, but not ZmMC9, are predominantly localized in a punctate distribution in both N. benthamiana and maize (Zea mays) protoplasts. Furthermore, the co-expression of ZmMC1 and ZmMC2 with Rp1-D21 and CCD21 causes their re-distribution from being uniformly distributed in the nucleocytoplasm to a punctate distribution co-localizing with ZmMC1 and ZmMC2. We reveal a novel role of plant MCs in modulating the NLR-mediated defense response and derive a model to explain it.

SummaryMacroorganisms’ genotypes shape their phenotypes, which in turn shape the habitat available to potential microbial symbionts. This influence of host genotype on microbiome composition has been demonstrated in many systems; however, most previous studies have either compared unrelated genotypes or delved into molecular mechanisms. As a result, it is currently unclear whether the heritability of host-associated microbiomes follows similar patterns to the heritability of other complex traits.We take a new approach to this question by comparing the microbiomes of diverse maize inbred lines and their F1hybrid offspring, which we quantified in both rhizosphere and leaves of field-grown plants using 16S-v4 and ITS1 amplicon sequencing.We show that inbred lines and hybrids differ consistently in composition of bacterial and fungal rhizosphere communities, as well as leaf-associated fungal communities. A wide range of microbiome features display heterosis within individual crosses, consistent with patterns for non-microbial maize phenotypes. For leaf microbiomes, these results were supported by the observation that broad-sense heritability in hybrids was substantially higher than narrow-sense heritability.Our results support our hypothesis that at least some heterotic host traits affect microbiome composition in maize.

A host-selective, proteinaceous maize toxin was identified from the culture filtrate of the maize pathogen Cochliobolus heterostrophus. A dominant gene for toxin susceptibility was identified on maize chromosome 4. A toxic activity was identified from the culture filtrate (CF) of the fungus Cochliobolus heterostrophus, causal agent of the maize disease southern leaf blight (SLB) with differential toxicity on maize lines. Two independent mapping populations; a 113-line recombinant inbred line population and a 258-line association population, were used to map loci associated with sensitivity to the CF at the seedling stage. A major QTL on chromosome 4 was identified at the same locus using both populations. Mapping in the association population defined a 400 kb region that contained the sensitivity locus. By comparing CF-sensitivity of the parents of the RIL population with that of the F1 progeny, we determined that the sensitivity allele was dominant. No relationship was observed between CF-sensitivity in seedlings and SLB susceptibility in mature plants; however, a significant correlation (− 0.58) was observed between SLB susceptibility and CF-sensitivity in seedlings. The activity of the CF was light-dependent and was sensitive to pronase, indicating that the toxin was proteinaceous.

Microbe associated molecular pattern (MAMPs) triggered immunity (MTI) is a key component of the plant innate immunity response to microbial recognition. However, most of our current knowledge of MTI comes from model plants (i.e., Arabidopsis thaliana) with comparatively less work done using crop plants. In this work, we studied the MAMP triggered oxidative burst (ROS) and the transcriptional response in two Sorghum bicolor genotypes, BTx623 and SC155-14E. SC155-14E is a line that shows high anthracnose resistance and the line BTx623 is susceptible to anthracnose. Our results revealed a clear variation in gene expression and ROS in response to either flagellin (flg22) or chitin elicitation between the two lines. While the transcriptional response to each MAMP and in each line was unique there was a considerable degree of overlap, and we were able to define a core set of genes associated with the sorghum MAMP transcriptional response. The GO term and KEGG pathway enrichment analysis discovered more immunity and pathogen resistance related DEGs in MAMP treated SC155-14E samples than in BTx623 with the same treatment. The results provide a baseline for future studies to investigate innate immunity pathways in sorghum, including efforts to enhance disease resistance.

In this study we mapped the QTL Qgls8 for gray leaf spot (GLS) resistance in maize to a ~130 kb region on chromosome 8 including five predicted genes. In previous work, using near isogenic line (NIL) populations in which segments of the teosinte (Zea mays ssp. parviglumis) genome had been introgressed into the background of the maize line B73, we had identified a QTL on chromosome 8, here called Qgls8, for gray leaf spot (GLS) resistance. We identified alternate teosinte alleles at this QTL, one conferring increased GLS resistance and one increased susceptibility relative to the B73 allele. Using segregating populations derived from NIL parents carrying these contrasting alleles, we were able to delimit the QTL region to a ~130 kb (based on the B73 genome) which encompassed five predicted genes.

Plants have the capacity to respond to conserved molecular features known as microbe-associated molecular patterns (MAMPs). The goal of this work was to assess variation in the MAMP response in sorghum, to map loci associated with this variation, and to investigate possible connections with variation in quantitative disease resistance. Using an assay that measures the production of reactive oxygen species, we assessed variation in the MAMP response in a sorghum association mapping population known as the sorghum conversion population (SCP). We identified consistent variation for the response to chitin and flg22—an epitope of flagellin. We identified two SNP loci associated with variation in the flg22 response and one with the chitin response. We also assessed resistance to Target Leaf Spot (TLS) disease caused by the necrotrophic fungus Bipolaris cookei in the SCP. We identified one strong association on chromosome 5 near a previously characterized disease resistance gene. A moderately significant correlation was observed between stronger flg22 response and lower TLS resistance. Possible reasons for this are discussed.

Summary The hypersensitive defence response is found in all higher plants and is characterized by a rapid cell death at the point of pathogen ingress. It is usually associated with pathogen resistance, though, in specific situations, it may have other consequences such as pathogen susceptibility, growth retardation and, over evolutionary timescales, speciation. Due to the potentially severe costs of inappropriate activation, plants employ multiple mechanisms to suppress inappropriate activation of HR and to constrain it after activation. The ubiquity of this response among higher plants despite its costs suggests that it is an extremely effective component of the plant immune system.

Background The hypersensitive defense response (HR) in plants is a fast, localized necrotic response around the point of pathogen ingress. HR is usually triggered by a pathogen recognition event mediated by a nucleotide-binding site, leucine-rich repeat (NLR) protein. The autoactive maize NLR gene Rp1-D21 confers a spontaneous HR response in the absence of pathogen recognition. Previous work identified a set of loci associated with variation in the strength of Rp1-D21-induced HR. A polygalacturonase gene homolog, here termed ZmPGH1, was identified as a possible causal gene at one of these loci on chromosome 7. Results Expression of ZmPGH1 inhibited the HR-inducing activity of both Rp1-D21 and that of another autoactive NLR, RPM1(D505V), in a Nicotiana benthamiana transient expression assay system. Overexpression of ZmPGH1 in a transposon insertion line of maize was associated with suppression of chemically-induced programmed cell death and with suppression of HR induced by Rp1-D21 in maize plants grown in the field. Conclusions ZmPGH1 functions as a suppressor of programmed cell death induced by at least two autoactive NLR proteins and by two chemical inducers. These findings deepen our understanding of the control of the HR in plants. Electronic supplementary material The online version of this article (10.1186/s12870-019-1897-5) contains supplementary material, which is available to authorized users.

Southern Leaf Blight, Northern Leaf Blight, and Gray Leaf Spot, caused by ascomycete fungi, are among the most important foliar diseases of maize worldwide. Previously, disease resistance quantitative trait loci (QTL) for all three diseases were identified in a connected set of chromosome segment substitution line (CSSL) populations designed for the identification of disease resistance QTL. Some QTL for different diseases co-localized, indicating the presence of multiple disease resistance (MDR) QTL. The goal of this study was to perform an independent test of several of the MDR QTL identified to confirm their existence and derive a more precise estimate of allele additive and dominance effects. Twelve F2:3 family populations were produced, in which selected QTL were segregating in an otherwise uniform genetic background. The populations were assessed for each of the three diseases in replicated trials and genotyped with markers previously associated with disease resistance. Pairwise phenotypic correlations across all the populations for resistance to the three diseases ranged from 0.2 to 0.3 and were all significant at the alpha level of 0.01. Of the 44 QTL tested, 16 were validated (identified at the same genomic location for the same disease or diseases) and several novel QTL/disease associations were found. Two MDR QTL were associated with resistance to all three diseases. This study identifies several potentially important MDR QTL and demonstrates the importance of independently evaluating QTL effects following their initial identification.

A protocol was developed for establishing embryogenic suspension cultures from in vitro-grown, thin shoot-tip sections of the banana cultivar Rasthali. The best medium for callus induction was an MS-based medium supplemented with 2 mg/l 2,4-D and 0.2 mg/l zeatin. The callus was transferred to liquid medium to establish embryogenic cell suspensions. These cultures were subsequently used for Agrobacterium-mediated transformation. The Agrobacterium tumefaciens strain EHA105 containing the binary vector pVGSUN with the als gene as a selectable marker and an intron-containing the gusA gene as a reporter gene was used for transformations. The herbicide Glean was used as a selection agent. Two hundred putative transformants were recovered, of which a set of 16 was tested by histochemical analysis for GUS expression and by Southern blot analysis with a probe for the gusA gene. The plants were positive for GUS expression and integration of the gusA gene. Two of the transformants were grown to maturity under greenhouse conditions. Bananas were harvested to test GUS expression by histochemical analysis. The fruit from both transgenics tested positive for GUS expression.

Physiological leaf spotting, or flecking, is a mild-lesion phenotype observed on the leaves of several commonly used maize (Zea mays) inbred lines and has been anecdotally linked to enhanced broad-spectrum disease resistance. Flecking was assessed in the maize nested association mapping (NAM) population, comprising 4,998 recombinant inbred lines from 25 biparental families, and in an association population, comprising 279 diverse maize inbreds. Joint family linkage analysis was conducted with 7,386 markers in the NAM population. Genome-wide association tests were performed with 26.5 million single-nucleotide polymorphisms (SNPs) in the NAM population and with 246,497 SNPs in the association population, resulting in the identification of 18 and three loci associated with variation in flecking, respectively. Many of the candidate genes colocalizing with associated SNPs are similar to genes that function in plant defense response via cell wall modification, salicylic acid- and jasmonic acid-dependent pathways, redox homeostasis, stress response, and vesicle trafficking/remodeling. Significant positive correlations were found between increased flecking, stronger defense response, increased disease resistance, and increased pest resistance. A nonlinear relationship with total kernel weight also was observed whereby lines with relatively high levels of flecking had, on average, lower total kernel weight. We present evidence suggesting that mild flecking could be used as a selection criterion for breeding programs trying to incorporate broad-spectrum disease resistance.

Quantitative resistance to maize common rust (causal agent Puccinia sorghi) was assessed in an association mapping population of 274 diverse inbred lines. Resistance to common rust was found to be moderately correlated with resistance to three other diseases and with the severity of the hypersensitive defense response previously assessed in the same population. Using a mixed linear model accounting for the confounding effects of population structure and flowering time, genome-wide association tests were performed based at 246,497 single-nucleotide polymorphism loci. Three loci associated with maize common rust resistance were identified. Candidate genes at each locus had predicted roles, mainly in cell wall modification. Other candidate genes included a resistance gene and a gene with a predicted role in regulating accumulation of reactive oxygen species.

The study of phenotypic variation in plant pathogenesis provides fundamental information about the nature of disease resistance. Cellular mechanisms that alter pathogenesis can be elucidated with confocal microscopy; however, systematic phenotyping platforms-from sample processing to image analysis-to investigate this do not exist. We have developed a platform for 3D phenotyping of cellular features underlying variation in disease development by fluorescence-specific resolution of host and pathogen interactions across time (4D). A confocal microscopy phenotyping platform compatible with different maize-fungal pathosystems (fungi: Setosphaeria turcica, Cochliobolus heterostrophus, and Cercospora zeae-maydis) was developed. Protocols and techniques were standardized for sample fixation, optical clearing, species-specific combinatorial fluorescence staining, multisample imaging, and image processing for investigation at the macroscale. The sample preparation methods presented here overcome challenges to fluorescence imaging such as specimen thickness and topography as well as physiological characteristics of the samples such as tissue autofluorescence and presence of cuticle. The resulting imaging techniques provide interesting qualitative and quantitative information not possible with conventional light or electron 2D imaging. Microsc. Res. Tech., 81:141-152, 2018. © 2016 Wiley Periodicals, Inc.

Disease resistance (R) genes encode nucleotide binding Leu-rich-repeat (NLR) proteins that confer resistance to specific pathogens. Upon pathogen recognition they trigger a defense response that usually includes a so-called hypersensitive response (HR), a rapid localized cell death at the site of pathogen infection. Intragenic recombination between two maize (Zea mays) NLRs, Rp1-D and Rp1-dp2, resulted in the formation of a hybrid NLR, Rp1-D21, which confers an autoactive HR in the absence of pathogen infection. From a previous quantitative trait loci and genome-wide association study, we identified genes encoding two key enzymes in lignin biosynthesis, hydroxycinnamoyltransferase (HCT) and caffeoyl CoA O-methyltransferase (CCoAOMT), adjacent to the nucleotide polymorphisms that were highly associated with variation in the severity of Rp1-D21-induced HR We have previously shown that the two maize HCT homologs suppress the HR conferred by Rp1-D21 in a heterologous system, very likely through physical interaction. Here, we show, similarly, that CCoAOMT2 suppresses the HR induced by either the full-length or by the N-terminal coiled-coil domain of Rp1-D21 also likely via physical interaction and that the metabolic activity of CCoAOMT2 is unlikely to be necessary for its role in suppressing HR. We also demonstrate that CCoAOMT2, HCTs, and Rp1 proteins can form in the same complexes. A model is derived to explain the roles of CCoAOMT and HCT in Rp1-mediated defense resistance.

The gene GRMZM2G318346 which encodes a cytochrome b–c1 complex subunit 7 is associated with variation in strength of the hypersensitive response in maize. We previously identified a QTL at 3,545,354 bp (B73 reference genome V2) on maize chromosome 5 associated with variation in the hypersensitive response (HR) conferred by the autoactive R-gene Rp1-D21 (Olukolu et al. in PLoS Genet 10:e1004562 2014). In this study, we show that a gene at this locus, GRMZM2G318346 which encodes a cytochrome b–c1 complex subunit seven (ZmQCR7), an important part of the mitochondrial electron transport chain, can suppress HR mediated by Rp1-D21 in a transient expression assay. ZmQCR7 alleles from two maize lines, W22 and B73 differ for the encoded proteins at just two sites, amino acid 27 (threonine and alanine in B73 and W22, respectively) and amino acid 109 (asparagine and serine), however, the B73 allele is much more effective at suppressing HR. We show that variation at amino acid 27 controlled this variation in HR-suppressing effects. We furthermore demonstrate that the B73 allele of ZmQCR7 can suppress HR induced by RPM1(D505 V), another autoactive R-gene, and that Arabidopsis homologs of ZmQCR7 can also suppress NLR-induced HR. The implications of these findings are discussed.

In this paper, we determine the genetic architecture controlling leaf flecking in maize and investigate its relationship to disease resistance and the defense response. Flecking is defined as a mild, often environmentally dependent lesion phenotype observed on the leaves of several commonly used maize inbred lines. Anecdotal evidence suggests a link between flecking and enhanced broad-spectrum disease resistance. Neither the genetic basis underlying flecking nor its possible relationship to disease resistance has been systematically evaluated. The commonly used maize inbred Mo17 has a mild flecking phenotype. The IBM-advanced intercross mapping population, derived from a cross between Mo17 and another commonly used inbred B73, has been used for mapping a number of traits in maize including several related to disease resistance. In this study, flecking was assessed in the IBM population over 6 environments. Several quantitative trait loci for flecking were identified, with the strongest one located on chromosome 6. Low but moderately significant correlations were observed between stronger flecking and higher disease resistance with respect to two diseases, southern leaf blight and northern leaf blight and between stronger flecking and a stronger defense response.

Field-grown maize is inoculated with Cochliobolus heterostrophus, causal agent of southern leaf blight disease, by dropping sorghum grains infested with the fungus into the whorl of each maize plant at an early stage of growth. The initial lesions produce secondary inoculum that is dispersed by wind and rain, causing multiple cycles of infection that assures a high uniform disease pressure over the entire field by the time of disease scoring, which occurs after anthesis. This method, with slight modifications, can also be used to study the maize fungal diseases northern leaf blight (caused by Exserohilum turcicum) and gray leaf spot (Cercospora zeae-maydis).

Plant diseases are responsible for substantial crop losses each year and pose a threat to global food security and agricultural sustainability. Improving crop resistance to pathogens through breeding is an environmentally sound method for managing disease and minimizing these losses. However, it is challenging to breed varieties with resistance that is effective, stable and broad-spectrum. Recent advances in genetic and genomic technologies have contributed to a better understanding of the complexity of host-pathogen interactions and have identified some of the genes and mechanisms that underlie resistance. This new knowledge is benefiting crop improvement through better-informed breeding strategies that utilize diverse forms of resistance at different scales, from the genome of a single plant to the plant varieties deployed across a region.

Alleles that confer multiple disease resistance (MDR) are valuable in crop improvement, although the molecular mechanisms underlying their functions remain largely unknown. A quantitative trait locus, qMdr9.02, associated with resistance to three important foliar maize diseases-southern leaf blight, gray leaf spot and northern leaf blight-has been identified on maize chromosome 9. Through fine-mapping, association analysis, expression analysis, insertional mutagenesis and transgenic validation, we demonstrate that ZmCCoAOMT2, which encodes a caffeoyl-CoA O-methyltransferase associated with the phenylpropanoid pathway and lignin production, is the gene within qMdr9.02 conferring quantitative resistance to both southern leaf blight and gray leaf spot. We suggest that resistance might be caused by allelic variation at the level of both gene expression and amino acid sequence, thus resulting in differences in levels of lignin and other metabolites of the phenylpropanoid pathway and regulation of programmed cell death.

Maize is the world's most produced crop, providing food, feed, and biofuel. Maize production is constantly threatened by the presence of devastating pathogens worldwide. Characterization of the genetic components underlying disease resistance is a major research area in maize which is highly relevant for resistance breeding programs. Quantitative disease resistance (QDR) is the type of resistance most widely used by maize breeders. The past decade has witnessed significant progress in fine-mapping and cloning of genes controlling QDR. The molecular mechanisms underlying QDR remain poorly understood and exploited. In this review we discuss recent advances in maize QDR research and strategy for resistance breeding.

In this study we examined the effects of two quantitative trait loci (QTL) for southern leaf blight (SLB) resistance on several agronomic traits including disease resistance and yield. B73–3B and B73–6A are two near-isogenic lines (NILs) in the background of the maize (Zea mays L.) inbred B73, each carrying one introgression (called 3B and 6A respectively) encompassing a QTL for SLB resistance. Sets of isohybrid triplets were developed by crossing B73, B73–3B, and B73–6A to several inbred lines. A subset of these triplets for which the B73–3B and/or B73–6A hybrid was significantly more SLB resistant than the B73 check hybrid was selected and assessed in multi-environment yield trials with and without disease. In the presence of SLB, 3B was associated with an approximately 3% yield increase over B73. 6A was associated with a yield advantage in the presence of SLB in specific pedigrees where the 6A resistance phenotype was highly expressed. Results suggested that both introgressions might confer a yield cost in the absence of SLB, but only introgression 6A was associated with a statistically significant reduction. We present evidence to suggest that the yield cost is associated with the resistance phenotype rather than with linkage drag. J.H. Santa-Cruz and P.J. Balint-Kurti, Dep. of Plant Pathology, N.C. State Univ., Raleigh, NC 27695; K.L. Kump, M.M. Goodman, M.D. Krakowsky, and J.B. Holland, Dep. of Crop Science, N.C. State Univ., Raleigh, NC 27695; C. Arellano, Dep. of Statistics, N.C. State Univ., Raleigh, NC 27695; M.D. Krakowsky, J.B. Holland, and P.J. BalintKurti, USDA-ARS, Plant Science Research Unit, Raleigh NC 27695. J.H. Santa-Cruz and K.L. Kump contributed equally to this work. Received 19 Aug. 2013. *Corresponding author (pjbalint@ncsu.edu ). Abbreviations: CL, NCSU Central Crops Research Station in Clayton, NC; CTAB, cetyltrimethylammonium bromide; dQTL, diseaseresistance quantitative trait locus (loci); DTA, days to anthesis; DTS, days to silking; KI, Cunningham Research Station in Kingston, NC; LE, Peanut Belt Research Station in Lewiston, NC; NIL, near isogenic line; PCR, polymerase chain reaction; SNP, single nucleotide polymorphism; QDR, quantitative disease resistance; QTL, quantitative trait locus (loci); R, resistance; sAUDPC, standardized area under the disease progress curve; SH, Sandhills Research Station in Jackson springs, NC; SLB, southern corn leaf blight; SSR, simple sequence repeat. Published in Crop Sci. 54:882–894 (2014). doi: 10.2135/cropsci2013.08.0553 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher. Published March 21, 2014