Disease Lesion Mimic Research Articles

Cloning the barley nec3 disease lesion mimic mutant using complementation by sequencing.

Disease lesion mimic (DLM) or necrotic mutants display necrotic lesions in the absence of pathogen infections. They can show improved resistance to some pathogens and their molecular dissection can contribute to revealing components of plant defense pathways. Although forward-genetics strategies to find genes causal to mutant phenotypes are available in crops, these strategies require the production of experimental cross populations, mutagenesis, or gene editing and are time- and resource-consuming or may have to deal with regulated plant materials. In this study, we described a collection of 34 DLM mutants in barley (Hordeum vulgare L.) and applied a novel method called complementation by sequencing (CBS), which enables the identification of the gene responsible for a mutant phenotype given the availability of two or more chemically mutagenized individuals showing the same phenotype. Complementation by sequencing relies on the feasibility to obtain all induced mutations present in chemical mutants and on the low probability that different individuals share the same mutated genes. By CBS, we identified a cytochrome P450 CYP71P1 gene as responsible for orange blotch DLM mutants, including the historical barley nec3 locus. By comparative phylogenetic analysis we showed that CYP71P1 gene family emerged early in angiosperm evolution but has been recurrently lost in some lineages including Arabidopsis thaliana (L.) Heynh. Complementation by sequencing is a straightforward cost-effective approach to clone genes controlling phenotypes in a chemically mutagenized collection. The TILLMore (TM) collection will be instrumental for understanding the molecular basis of DLM phenotypes and to contribute knowledge about mechanisms of host-pathogen interaction.

Mutations in a barley cytochrome P450 gene enhances pathogen induced programmed cell death and cutin layer instability.

Disease lesion mimic mutants (DLMMs) are characterized by the spontaneous development of necrotic spots with various phenotypes designated as necrotic (nec) mutants in barley. The nec mutants were traditionally considered to have aberrant regulation of programmed cell death (PCD) pathways, which have roles in plant immunity and development. Most barley nec3 mutants express cream to orange necrotic lesions contrasting them from typical spontaneous DLMMs that develop dark pigmented lesions indicative of serotonin/phenolics deposition. Barley nec3 mutants grown under sterile conditions did not exhibit necrotic phenotypes until inoculated with adapted pathogens, suggesting that they are not typical DLMMs. The F2 progeny of a cross between nec3-γ1 and variety Quest segregated as a single recessive susceptibility gene post-inoculation with Bipolaris sorokiniana, the causal agent of the disease spot blotch. Nec3 was genetically delimited to 0.14 cM representing 16.5 megabases of physical sequence containing 149 annotated high confidence genes. RNAseq and comparative analysis of the wild type and five independent nec3 mutants identified a single candidate cytochrome P450 gene (HORVU.MOREX.r2.6HG0460850) that was validated as nec3 by independent mutations that result in predicted nonfunctional proteins. Histology studies determined that nec3 mutants had an unstable cutin layer that disrupted normal Bipolaris sorokiniana germ tube development.

Commonly and Specifically Activated Defense Responses in Maize Disease Lesion Mimic Mutants Revealed by Integrated Transcriptomics and Metabolomics Analysis.

Disease lesion mimic (Les/les) mutants display disease-like spontaneous lesions in the absence of pathogen infection, implying the constitutive activation of defense responses. However, the genetic and biochemical bases underlying the activated defense responses in those mutants remain largely unknown. Here, we performed integrated transcriptomics and metabolomics analysis on three typical maize Les mutants Les4, Les10, and Les17 with large, medium, and small lesion size, respectively, thereby dissecting the activated defense responses at the transcriptional and metabolomic level. A total of 1,714, 4,887, and 1,625 differentially expressed genes (DEGs) were identified in Les4, Les10, and Les17, respectively. Among them, 570, 3,299, and 447 specific differentially expressed genes (SGs) were identified, implying a specific function of each LES gene. In addition, 480 common differentially expressed genes (CGs) and 42 common differentially accumulated metabolites (CMs) were identified in all Les mutants, suggesting the robust activation of shared signaling pathways. Intriguingly, substantial analysis of the CGs indicated that genes involved in the programmed cell death, defense responses, and phenylpropanoid and terpenoid biosynthesis were most commonly activated. Genes involved in photosynthetic biosynthesis, however, were generally repressed. Consistently, the dominant CMs identified were phenylpropanoids and flavonoids. In particular, lignin, the phenylpropanoid-based polymer, was significantly increased in all three mutants. These data collectively imply that transcriptional activation of defense-related gene expression; increase of phenylpropanoid, lignin, flavonoid, and terpenoid biosynthesis; and inhibition of photosynthesis are generalnatures associated with the lesion formation and constitutively activated defense responses in those mutants. Further studies on the identified SGs and CGs will shed new light on the function of each LES gene as well as the regulatory network of defense responses in maize.

Extracting complex lesion phenotypes in Zea mays

Complex phenotypes are of growing importance in agriculture and medicine. In Zea mays, the most widely produced crop in the world (United States Department of Agriculture. World Agricultural Production. United States Department of Agriculture, Foreign Agricultural Service, Washington, 2015), the disease lesion mimic mutants produce regions of discolored or necrotic tissue in otherwise healthy plants. These mutants are of particular interest due to their apparent action on immune response pathways, providing insight into how plants protect against infectious agents. These phenotypes vary considerably as a function of genotype and environmental conditions, making them a rich, though challenging, phenotypic problem. To segment and quantitate these lesions, we present a novel cascade of adaptive algorithms able to accurately segment the diversity of Z. mays lesions. First, multiresolution analysis of the image allows for salient features to be detected at multiple scales. Next, gradient vector diffusion enhances relevant gradient vectors while suppressing noise. Finally, an active contour algorithm refines the lesion boundary, producing a final segmentation for each lesion. We compare the results from this cascade with manual segmentations from human observers, demonstrating that our algorithm is comparable to humans while having the potential to speed analysis by several orders of magnitude.

Inheritance of dlm and ti genes in Soybean

dlm mutant newly identified displays necrotic spots with chlorotic halo despite the absence of any detectable pathogens in soybean. Kunitz trypsin inhibitor (KTI) protein of mature soybean seed is a main anti-nutritional factor and is controlled by Ti allele in soybean seed. The objective of this research was to determine the linkage or independent assortment between dlm allele and ti allele. F2 population was made by crossing between Gaechuck2ho (DlmDlmtiti) and T363 (dlmdlmTiTi) parents. The normal leaf or disease lesion mimic leaf trait of parents and F2 individual plants was recorded at R2 and R3 growth stage from field. The random seeds from individual F2 plants harvested were analysed electrophoretically to determine the presence of the KTI protein. The disease lesion mimic trait was matched to 3 : 1 ratio at the 0.01 levels of probability. The absence of the KTI protein was controlled by a single recessive gene. In F2 generation, the ratios of 102 Ti_Dlm_: 21 Ti_dlmdlm : 24 titiDlm_ : 4 titidlmdlm were observed. Segregation ratio gave a good fit to the 9 : 3 : 3 : 1 ratio based on F2 generation at the 0.01 levels of probability. The data confirmed that dlm allele controlling disease lesion mimic trait was inherited independently with the ti allele controlling absence of KTI protein in soybean.

Barley necrotic locus nec1 encodes the cyclic nucleotide-gated ion channel 4 homologous to the Arabidopsis HLM1

Barley homolog of the Arabidopsis necrotic (disease lesion mimic) mutant HLM1 that encodes the cyclic nucleotide-gated ion channel 4 was cloned. Barley gene was mapped genetically to the known necrotic locus nec1 and subsequent sequence analysis identified mutations in five available nec1 alleles confirming barley homolog of Arabidopsis HLM1 as the NEC1 gene. Two fast neutron (FN) induced mutants had extensive deletions in the gene, while two previously described nec1 alleles had either a STOP codon in exon 1 or a MITE insertion in intron 2 which caused alternative splicing, frame shift and production of a predicted non-functional protein. The MITE insertion was consistent with the reported spontaneous origin of the nec1 Parkland allele. The third FN mutant had a point mutation in the coding sequence which resulted in an amino acid change in the conserved predicted cyclic nucleotide-gated ion channel pore region. The expression of two pathogenesis-related genes, HvPR-1a and beta-1,3-glucanase, was elevated in two FN necrotic lines. Ten other members of the barley cyclic nucleotide-gated ion channel gene family were identified and their position on barley linkage map is reported.

The Phenotype of the Soybean Disease-Lesion Mimic (dlm) Mutant is Light-Dependent and Associated with Chloroplast Function

The dlm (disease lesion mimic) mutant of soybean (Glycine max L. Merr) shows the similar lesion of a soybean disease caused by a fungus, Corynespora cassilcola. The lesion was examined at cellular and molecular level. Trypan blue staining result indicated that cell death was detectable in the entire region of leaves excluding veins when the lesions had already been developed. We found that the mesophyll cells of palisade layer in the dim mutant appeared to be wider apart from each other. The chloroplasts of the dim mutant cells contained bigger starch granules than those in normal plants. We also found that the lesion development of dlm plant was light-dependent and the starch degradation during the dark period of diurnal cycle was impaired in the mutant. Three soybean pathogenesis-related genes, PR-1a, PR-4, and PR-10, were examined for their expression patterns during the development of disease lesion mimic. The expression of all three genes was up-regulated to some extent upon the appearance of the disease lesion mimic. Although the exact function of DLM protein remains elusive, our data would provide some insight into mechanism underling the cell death associated with the dim mutation.

Barley putative hypersensitive induced reaction genes: genetic mapping, sequence analyses and differential expression in disease lesion mimic mutants.

The hypersensitive response (HR) is one of the most-efficient forms of plant defense against biotrophic pathogens, and results in localized cell death and the formation of necrotic lesions; however, the molecular components of pathways leading to HR remain largely unknown. Barley ( Hordeum vulgare ssp. vulgare L.) cDNAs for putative hypersensitive-induced reaction ( HIR) genes were isolated based on DNA and amino-acid homologies to maize HIR genes. Analyses of the cDNA and genomic sequences and genetic mapping found four distinct barley HIR genes, Hv-hir1, Hv-hir2, Hv-hir3 and Hv-hir4, on chromosomes 4(4H) bin10, 7(5H) bin04, 7(5H) bin07 and 1(7H) bin03, respectively. Hv-hir1, Hv-hir2 and Hv-hir3 genes were highly homologous at both DNA and the deduced amino-acid level, but the Hv-hir4 gene was similar to the other genes only at the amino-acid sequence level. Amino-acid sequence analyses of the barley HIR proteins indicated the presence of the SPFH protein-domain characteristic for the prohibitins and stomatins which are involved in control of the cell cycle and ion channels, as well as in other membrane-associated proteins from bacteria, plants and animals. HIR genes were expressed in all organs and developement stages analyzed, indicating a vital and non-redundant function. Barley fast-neutron mutants exhibiting spontaneous HR (disease lesion mimic mutants) showed up to a 35-fold increase in Hv-hir3 expression, implicating HIR genes in the induction of HR.

A maize defense-inducible gene is a major facilitator superfamily member related to bacterial multidrug resistance efflux antiporters.

A defense-inducible maize gene was discovered through global mRNA profiling analysis. Its mRNA expression is induced by pathogens and defense-related conditions in various tissues involving both resistant and susceptible interactions. These include Cochliobolus heterostrophus and Cochliobolus carbonum infection, ultraviolet light treatment, the Les9 disease lesion mimic background, and plant tissues engineered to express flavonoids or the avirulence gene avrRxv. The gene was named Zm-mfs1 after it was found to encode a protein related to the major facilitator superfamily (MFS) of intregral membrane permeases. It is most closely related to the bacterial multidrug efflux protein family, typified by the Escherichia coli TetA, which are proton motive force antiporters that export antimicrobial drugs and other compounds, but which can be also involved in potassium export/proton import or potassium re-uptake. Other related plant gene sequences in maize, rice, and Arabidopsis were identified, three of which are introduced here. Among this new plant MFS subfamily, the characteristic MFS motif in cytoplasmic TM2-TM3 loop, and the antiporter family motif in transmembrane domain TM5 are both conserved, however the TM7 and the cytoplasmic TM8-TM9 loop are divergent from those of the bacterial multidrug transporters. We hypothesize that Zm-Mfs1 is a prototype of a new class of plant defense-related proteins that could be involved in either of three nonexclusive roles: (1) export of antimicrobial compounds produced by plant pathogens; (2) export of plant-generated antimicrobial compounds; and (3) potassium export and/or re-uptake, as can occur in plant defense reactions.

Inheritance of disease lesion mimic leaf trait in groundnut.

In groundnut, two identical mutants with disease lesion mimic leaf trait were isolated independently from two different parents through induced mutagenesis and in vitro culture technique. The leaf chlorophyll content in both the mutants was found to be drastically reduced. The segregation pattern in the F(2) and F(3) generations for normal and mutant traits fitted a 13:3 ratio, indicating that the disease lesion mimic trait in the mutants was due to suppressive gene action. Both mutants were allelic for the disease mimic trait.

A humidity-sensitive Arabidopsis copine mutant exhibits precocious cell death and increased disease resistance.

The copines are a newly identified class of calcium-dependent, phospholipid binding proteins that are present in a wide range of organisms, including Paramecium, plants, Caenorhabditis elegans, mouse, and human. However, the biological functions of the copines are unknown. Here, we describe a humidity-sensitive copine mutant in Arabidopsis. Under nonpermissive, low-humidity conditions, the cpn1-1 mutant displayed aberrant regulation of cell death that included a lesion mimic phenotype and an accelerated hypersensitive response (HR). However, the HR in cpn1-1 showed no increase in sensitivity to low pathogen titers. Low-humidity-grown cpn1-1 mutants also exhibited morphological abnormalities, increased resistance to virulent strains of Pseudomonas syringae and Peronospora parasitica, and constitutive expression of pathogenesis-related (PR) genes. Growth of cpn1-1 under permissive, high-humidity conditions abolished the increased disease resistance, lesion mimic, and morphological mutant phenotypes but only partially alleviated the accelerated HR and constitutive PR gene expression phenotypes. The disease resistance phenotype of cpn1-1 suggests that the CPN1 gene regulates defense responses. Alternatively, the primary function of CPN1 may be the regulation of plant responses to low humidity, and the effect of the cpn1-1 mutation on disease resistance may be indirect.

Genetical and anatomical analyses of a leaf flecking mutant in Triticum aestivum L.

Flecking trait in the mutant C591 (M8) Triticum aestivum L. is a stable,developmentally programmed, dominant mutation under monogenic control resembling pathogenic attack and starts appearing only from boot leaf stage of the plant. Mutant plants differ significantly from normal plants in terms of total chlorphyll contents only at later stages of symptom spread when the flecks fully cover the leaf sheath and leaves. However, total grain weight per main spike of mutant did not differ significantly from the normal plants. Microscopic studies of the mutant leaves did not reveal any damaging effect of the mutation on leaf anatomy per se, even though differences were observed in chlorophyll filling in mesophyll cells. Considering the peculiar characteristics of the mutation, many of which resembling the disease lesion mimic mutations in other crops, this is suspected to be such a mutation in wheat.

Prohibitins, Stomatins, and Plant Disease Response Genes Compose a Protein Superfamily That Controls Cell Proliferation, Ion Channel Regulation, and Death

Prohibitins, stomatins, and a group of plant defense response genes are demonstrated to belong to a novel protein superfamily. This superfamily is bound by similar primary and secondary predicted protein structures and hydropathy profiles. A PROSITE-formatted regular expression was generated that is highly predictive for identifying members of this superfamily using PHI-BLAST. The superfamily is named PID (proliferation, ion, and death) because prohibitins are involved in proliferation and cell cycle control, stomatins are involved in ion channel regulation, and the plant defense-related genes are involved in cell death. The plant defense gene family is named HIR (hypersensitive induced reaction) because its members are associated with hypersensitive reactions involving cell death and pathogen resistance. For this study, eight novel maize genes were introduced: four closely related to prohibitins (Zm-phb1, Zm-phb2, Zm-phb3, and Zm-phb4), one to stomatins (Zm-stm1), and three to a gene implicated in plant disease responses (Zm-hir1, Zm-hir2, and Zm-hir3). The maize Zm-hir3 gene transcript is up-regulated in a disease lesion mimic mutation (Les9), supporting a role in maize defense responses. Members of this gene superfamily are involved in diverse functions, but their structural similarity suggests a conserved molecular mechanism, which we postulate to be ion channel regulation.

Genes for resistance to northern corn leaf blight in diverse maize populations

AbstractTurcicum or northern corn leaf blight (NCLB) incited by the ascomycete Setosphaeria turcica, anamorph Exserohilum turcicum, is a ubiquitous foliar disease of maize. Diverse sources of qualitative and quantitative resistance are available but qualitative resistances (Ht genes) are often unstable. In the tropics especially, they are either overcome by new virulent races or they suffer from climatically sensitive expression. Quantitative resistance is expressed independently of the physical environment and has never succumbed to S. turcica pathotypes in the field. This review emphasizes the identification and mapping of genes related to quantitative NCLB resistance. We deal with the consistency of the genomic positions of quantitative trait loci (QTL) controlling resistance across different maize populations, and with the clustering of genes for resistance to S. turcica and other fungal pathogens or insect pests in the maize genome. Implications from these findings for further genomic research and resistance breeding are drawn.Incubation period (IP) and area under the disease progress curve (AUDPC), based on multiple disease ratings, are important component traits of quantitative NCLB resistance. They are generally tightly correlated (rp≅ 0.8) and highly heritable (h2≅ 0.75). QTL for resistance to NCLB (IP and AUDPC) were identified and characterized in three mapping populations (A, B, C). Population A, a set of 121‐150 F3 families of the cross B52×mo17, represented US Corn Belt germplasm with a moderate level of resistance. It was field‐tested in Iowa, USA, and Kenya, and genotyped at 112 restriction fragment length polymorphism (RFLP) loci. Population B consisted of 194‐256 F3 families of the cross Lo951×CML202, the first parent being a Corn‐Belt‐derived European inbred line and the second parent being a highly resistant tropical African inbred line. The population was also tested in Kenya and genotyped with 110 RFLP markers. Population C was derived from a cross between two early‐maturing European inbred lines, D32 and D145, both having a moderate level of resistance. A total of 220 F3 families were tested in Switzerland and characterized with 87 RFLP and seven SSR markers. In each of the three studies, 12‐13 QTL were detected by composite interval mapping at a signifcance threshold of LOD=2.5. The phenotypic and the genotypic variance were explained to an extent of 50‐70% and 60‐80%, respectively. Gene action was additive to partly dominant, as in previous generation means and combining ability analyses with other genetic material. In each population, gene effects of the QTL were of similar magnitude and no putative major genes were discovered. QTL for AUDPC were located on chromosomes 1 to 9. All three populations carried QTL in identical genomic regions on chromosomes 3 (bin 3.06/07), 5 (bin 3.06/07) and 8 (bin 8.05/06). The major genes Ht2 and Htn1 were also mapped to bins 8.05 and 8.06, suggesting the presence of a cluster of closely linked major and minor genes. The chromosomal bins 3.05, 5.04 and 8.05, or adjacent intervals, were further associated with QTL and major genes for resistance to eight other fungal diseases and insect pests of maize. Bins 1.05/07 and 9.05 were found to carry population‐specifc genes for resistance to S. turcica and other organisms. Several disease lesion mimic mutations, resistance gene analogues and genes encoding pathogenesis‐related proteins were mapped to regions harbouring NCLB resistance QTL.

The yeast polyadenylate-binding protein (PAB1) gene acts as a disease lesion mimic gene when expressed in plants.

We have expressed the gene (PAB1) encoding the yeast polyadenylate-binding protein (Pab1p) in tobacco. Plants that accumulate the Pab1p display a range of abnormalities, ranging from a characteristic chlorosis in leaves to a necrosis and large inhibition of growth. The severity of these abnormalities reflects the levels of yeast Pab1p expression in the transgenic plants. In contrast, no obvious differences could be seen in callus cultures between the transgene and vector control. Plants that display PAB-associated abnormalities were resistant to a range of plant pathogens, and had elevated levels of expression of a pathogenesis-related gene. These two properties--impairment of growth and induction of defense responses--indicate that the yeast PAB1 gene can act as a disease lesion mimic gene in plants.

Molecular characterization of the maize Rp1-D rust resistance haplotype and its mutants.

The Rp1-D gene for resistance to maize common rust (Puccinia sorghi) is a member of a complex locus (haplotype) composed of Rp1-D and approximately eight other gene homologs. The identity of Rp1-D was demonstrated by using two independent gene-tagging approaches with the transposons Mutator and Dissociation. PIC20, a disease resistance (R) gene analog probe previously mapped to the rp1 locus, detected insertion of Dissociation in an Rp1-D mutation and excision in three revertants. Independent libraries probed with the PIC20 or Mutator probes resulted in isolation of the same gene sequence. Rp1-D belongs to the nucleotide binding site, leucine-rich repeat class of R genes. However, unlike the rust resistance genes M and L6 from flax, the maize Rp1-D gene does not encode an N-terminal domain with similarity to the signal transduction domains of the Drosophila Toll protein and mammalian interleukin-1 receptor. Although the abundance of transcripts of genes from the rp1 complex changed with leaf age, there was no evidence of any change due to inoculation with avirulent or virulent rust biotypes. A set of 27 Rp1-D mutants displayed at least nine different deletions of Rp1-D gene family members that were consistent with unequal crossing-over events. One mutation (Rp1-D*-24) resulted in deletion of all but one gene family member. Other unique deletions were observed in the disease lesion mimic Rp1-D*-21 and the partially susceptible mutant Rp1-D*-5. Different rp1 specificities have distinct DNA fingerprints (haplotypes). Analysis of recombinants between rp1 specificities indicated that recombination had occurred within the rp1 gene complex. Similar analyses indicated that the rust R genes at the rp5 locus, 2 centimorgans distal to rp1, are not closely related to Rp1-D.

Brief communication. Inheritance of a disease lesion mimic mutant in soybean

Brief communication. Inheritance of a disease lesion mimic mutant in soybean

Partial Resistance to Root-Borne Infection by Phytophthora sojae in Three Allelic Necrotic Root Mutants in Soybean

Disease lesion mimic mutations that affect aerial parts of plants have been characterized in several species, but root disease mimics have not been previously described. Our objective was to characterize genetically and physiologically three independently derived necrotic root mutants found in soybean. The three mutants were inherited as a single recessive gene, were allelic, and were not linked to any of seven marker traits tested. When inoculated via the hypocotyl, necrotic root plants were susceptible to hyphal infection by a compatible race of the fungal pathogen Phytophthora sojae. In contrast, the roots showed partial resistance to infection by P. sojae zoospores, which correlated with the accumulation of isoflavonoid phytoalexins and group 2 peroxidases under axenic conditions. However, activation of the defense response in roots of the necrotic root mutants was insufficient to prevent nodulation by the symbiotic microorganism Bradyrhizobium japonicum.

Systemic acquired resistance signal transduction

Abstract Systemic acquired resistance (SAR) is an inducible plant defense response in which a prior foliar pathogen infection activates resistance in noninfected foliar tissues. Salicylic acid (SA) accumulation is essential for the establishment of SAR. While SA is probably not the long‐distance systemic signal instrumental for SAR activation, it is required for transduction of the signal in noninfected tissues. Although SAR was first described as a response to necrogenic pathogen infection, synthetic chemicals have been identified that effectively activate SAR. Elucidation of SAR signal transduction has been facilitated by the identification and characterization of Arabidopsis mutants. Disease lesion mimic mutants exhibit constitutive SAR as well as spontaneous lesion formation similar to pathogen‐associated hypersensitive cell death. Some disease lesion mimic mutants do not exhibit a lesioned phenotype when SA accumulation is prevented, thereby providing evidence for a feedback loop in SAR signal transd...

Disease lesion mimics of maize: A model for cell death in plants

AbstractA class of maize mutants, collectively known as disease lesion mimics, display discrete disease‐like symptoms in the absence of pathogens. It is intriguing that a majority of these lesion mimics behave as dominant gain‐of‐function mutations. The production of lesions is strongly influenced by light, temperature, developmental state and genetic background. Presently, the biological significance of this lesion mimicry is not clear, although suggestions have been made that they may represent defects in the plants' recognition of, or response to, pathogens. One feature that is common to all lesion mimics is their association with cell death. In plants, as in animals, a number of developmental and pathological processes exist where controlled cell death, whether programmed or triggered in response to physiological or environmental stimuli, constitutes the normal aspect of life. Might disease lesion mimic mutations represent variants where regulation of desirable cell death has gone awry? In this paper we argue that this might be the case, and further conjecture that these mutants offer a unique opportunity for studying the genetic and cellular mechanisms of cell death in plants.