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PubMed2005

Redox control of systemic acquired resistance.

Changes in gene expression during systemic acquired resistance (SAR) require the phenolic signaling molecule salicylic acid (SA) and are modulated by the interaction between the NON EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) protein and members of the TGA family of transcription factors. In the past two years, the activities of NPR1 and of the TGA factors TGA1 and TGA4 have been shown to be modulated by SA-induced oxidoreduction (redox) modifications of key cysteine residues. Reduction of two conserved cysteines in NPR1 leads to its monomerization and nuclear localization, which is required for the activation of PATHOGENESIS-RELATED(PR) genes. Reduction of conserved cysteines in TGA1 and TGA4 enables their interaction with NPR1, which acts as a redox-sensitive cofactor in stimulating TGA1 DNA-binding activity. The identity of the redox mediators that are involved in regulating NPR1 and TGA factors is unknown. However, a novel thioredoxin interacts with the disease resistance protein Cf-9 and modulates Cf-9-dependent signaling. These results begin to provide a mechanistic understanding of how SAR is regulated by redox conditions.

Gene Expression Regulation, Plant↗

PubMed2004

Tandem and segmental gene duplication and recombination in the evolution of plant disease resistance gene.

NBS-LRR genes are the major class of disease resistance genes in flowering plants, and are arranged as single genes and as clustered loci. The evolution of these genes has been investigated in Arabidopsis thaliana by combining data on their genomic organisation and position in phylogenetic trees. Tandem and segmental duplications distribute and separate NBS-LRR genes in the genome. It is, however, unclear by which mechanism(s) NBS-LRR genes from different clades are sampled into heterogeneous clusters. Once physically removed from their closest relatives, the NBS-LRR genes might adopt and preserve new specificities because they are less prone to sequence homogenization.

Evolution, Molecular↗

PubMed1999

Characterization of plant nematode genes: identifying targets for a transgenic defence.

Current control of plant parasitic nematodes often relies on highly toxic and environmentally harmful nematicides. As their use becomes increasingly restricted there is an urgent need to develop crop varieties with resistance to nematodes. The limitations surrounding conventional plant breeding ensure there is a clear opportunity for transgenic resistance to lessen current dependence on chemical control. The increasing use of molecular biology techniques in the field of plant nematology is now providing useful information for the design of novel defences to meet the new needs. Plant responses to parasitism are being investigated at the molecular level and nematode gene products that could be targets for a direct anti-nematode defence are being characterized. The potential of an anti-feedant approach to nematode control has been demonstrated. It is based on the transgenic expression of proteinase inhibitors. The rational development of this strategy involves characterization of nematode proteinase genes and optimization of inhibitors by protein engineering. Durability of the resistance can be enhanced by stacking transgenes directed at different nematode targets.

Animals↗

PubMed1997

Cloning and characterisation of a gene encoding an antiviral protein from Clerodendrum aculeatum L.

The Clerodendrum aculeatum-systemic resistance inducing (CA-SRI) protein, a 34 kDa basic protein, plays a key role in inducing strong systemic resistance in susceptible plants against various plant viruses [22]. We have cloned the cDNA encoding the CA-SRI from C. aculeatum leaves using antibodies raised against the purified protein and degenerate oligonucleotide probes derived from microsequencing of the CA-SRI protein. The full-length cDNA consisted of 1218 nucleotides with an open reading frame of 906 bp. The deduced amino acid sequence of CA-SRI protein showed varying homology (ranging from 11 to 54%) to the ribosome inactivating proteins (RIPs) from other plant species. CA-SRI inhibited in vitro protein synthesis both in rabbit reticulocyte lysate and wheat germ lysate but not in Escherichia coli in vitro translation system. The CA-SRI open reading frame was expressed in an E. coli expression vector and the purified recombinant protein inhibited protein synthesis in rabbit reticulocyte lysate. Southern blot analysis indicated that the CA-SRI gene may be present in low copy number.

Amino Acid Sequence↗

PubMed1999

Rice calcium-dependent protein kinase isoforms OsCDPK2 and OsCDPK11 show different responses to light and different expression patterns during seed development.

We investigated the spatial and temporal expression patterns of two rice calcium-dependent protein kinases (CDPKs), OsCDPK2 and OSCDPK11, using isoform-specific antisera. Bands of the expected molecular sizes for OsCDPK2 (59 kDa) and OsCDPK11 (61 kDa) were detected on western blots. OsCDPK2 and OsCDPK11 mRNA and protein levels increased in unison during flower development. However, at the onset of seed development, the protein expression profiles diverged significantly. OsCDPK2 protein was expressed at low levels during early seed development, but increased to high levels that were maintained in later stages (20 days after fertilisation, DAF). Conversely, OsCDPK11 protein levels were high at the beginning of seed development, but fell rapidly from 10 DAF onwards. This decrease in the level of OsCDPK11 protein was associated with the abundant synthesis of a truncated mRNA species. OsCDPK2 expression was also closely associated with light perception. OsCDPK2 protein was barely detectable in green leaves exposed to light, but levels increased sharply when plants were shifted to darkness. Initially, this increase reflected a rapid elevation in the levels of OsCDPK2 mRNA, which was normally located in the mesophyll. Conversely, OsCDPK11 mRNA and protein levels were unaffected by light. These data strongly indicate that two rice CDPK isoforms have different functions in seed development and in response to light in leaves.

Blotting, Western↗

PubMed2002

Characterization of a stamen-specific cDNA encoding a novel plant defensin in Chinese cabbage.

We isolated a stamen-specific cDNA, BSD1 (Brassica stamen specific plant defensin 1) that encodes a novel plant defensin peptide in Chinese cabbage (Brassica campestris L. ssp. pekinensis). Plant defensins are antimicrobial peptides containing eight highly conserved cysteine residues linked by disulfide bridges. In BSD1, the eight cysteine residues and a glutamate residue at position 29 are conserved whereas other amino acid residues of the plant defensins consensus sequence are substituted. BSD1 transcripts accumulate specifically in the stamen of developing flowers and its level drops as the flowers mature. The recombinant BSD1 produced in Escherichia coli showed antifungal activity against several phytopathogenic fungi. Furthermore, constitutive over-expression of the BSD1 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter conferred enhanced tolerance against the Phytophthora parasitica in the transgenic tobacco plants.

Amino Acid Sequence↗

PubMed2003

Sporamin-mediated resistance to beet cyst nematodes (Heterodera schachtii Schm.) is dependent on trypsin inhibitory activity in sugar beet (Beta vulgaris L.) hairy roots.

Sporamin, a sweet potato tuberous storage protein, is a Kunitz-type trypsin inhibitor. Its capability of conferring insect-resistance on transgenic tobacco and cauliflower has been confirmed. To test its potential as an anti-feedant for the beet cyst nematode (Heterodera schachtii Schm.), the sporamin gene SpTI-1 was introduced into sugar beet (Beta vulgaris L.) by Agrobacterium rhizogenes-mediated transformation. Twelve different hairy root clones expressing sporamin were selected for studying nematode development. Of these, 8 hairy root clones were found to show significant efficiency in inhibiting the growth and development of the female nematodes whereas 4 root clones did not show any inhibitory effects even though the SpTI-1 gene was regularly expressed in all of the tested hairy roots as revealed by northern and western analyses. Inhibition of nematode development correlated with trypsin inhibitor activity but not with the amount of sporamin expressed in hairy roots. These data demonstrate that the trypsin inhibitor activity is the critical factor for inhibiting growth and development of cyst nematodes in sugar beet hairy roots expressing the sporamin gene. Hence, the sweet potato sporamin can be used as a new and effective anti-feedant for controlling cyst nematodes offering an alternative strategy for establishing nematode resistance in crops.

Animals↗

PubMed2003

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.

Amino Acid Sequence↗

PubMed2003

Rapid induction of a protein disulfide isomerase and defense-related genes in wheat in response to the hemibiotrophic fungal pathogen Mycosphaerella graminicola.

Mycosphaerella graminicola, incitant of septoria tritici blotch, is a widespread and significant pathogen of wheat that is not closely related to other fungi being developed as genetic models for host-pathogen interactions. Several resistance genes in wheat have been identified, yet the molecular mechanisms of resistance are unknown. To identify host genes involved in the resistance response, expression profiles of the wheat line Tadinia (containing the Stb4 gene for resistance) and the susceptible line Yecora Rojo, non-inoculated or inoculated with M. graminicola, were compared by differential-display polymerase chain reaction (DD-PCR). Among the differentially expressed genes was a protein disulfide isomerase (PDI), which is well known as a molecular chaperone and component of signal-transduction pathways in animal systems but had not been implicated previously in plant defense response. Real-time quantitative reverse-transcription PCR and northern analysis revealed that PDI was induced within 3 h of inoculation with highest induction in the pathogen-treated resistant lines. These responses of PDI were similar to the early and strong resistance-related responses displayed by the pathogenesis-related (PR) proteins, PR-1, PR-2 and PR-5. In contrast, a wheat lipoxygenase was down-regulated in the resistant lines at time points corresponding with peak induction of the PR genes. Thus, part of the resistance mechanism may involve repression of a gene that could otherwise aid fungal growth. Wheat responds much more rapidly than believed previously to signals produced by M.graminicola. These early responses begin prior to penetration of the host and appear to determine the outcome of the host-pathogen interaction.

Ascomycota↗

PubMed1997

Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat.

More than 100 resistance genes against wheat rust pathogens have been described in wheat and its relatives. Although many of them have been extensively used in wheat resistance breeding, none of these resistance loci has yet been analyzed at the molecular level. By screening a set of near-isogenic lines carrying different leaf rust resistance genes with a wheat probe encoding a serine/ threonine protein kinase, we detected a polymorphic DNA fragment in the line with the Lr10 resistance gene. This fragment mapped to the Lr10 disease resistance locus and encodes a receptor-like protein kinase which we called LRK10. LRK10 contains a new type of extracellular domain not found in known plant or animal receptor kinases. Several conserved amino acids in S-domain glycoproteins and receptor-like kinases were also found in LRK10, suggesting that LRK10 and S-domain proteins belong to the same superfamily of specific recognition molecules in plants. Lrk10 was expressed at low levels in young seedlings and belongs to a gene family. Analysis of wheat lines with and without the Lr10 gene demonstrated that Lrk10 and Lr10 belong to the same genetic locus. We conclude that gene isolation based on protein kinase homology can identify new receptor domains and provide candidates for disease resistance genes in the complex wheat genome.

Alleles↗

PubMed1998

Disease resistance gene homologs correlate with disease resistance loci of Arabidopsis thaliana.

The disease resistance genes RPS2 of Arabidopsis and N of tobacco, among other recently cloned resistance genes, share several conserved sequences. Degenerate oligonucleotide primers, based on conserved sequences in the nucleotide binding site (NBS) and a weak hydrophobic domain of RPS2 and N, were used to amplify homologous sequences from Arabidopsis thaliana. Amplification products were obtained that were similar in sequence to the disease resistance genes RPS2, RPM1, N and L6. The Arabidopsis CIC-YAC library was used to identify the position of the disease resistance homologs on the Arabidopsis genome. Their map positions could be correlated with the disease resistance loci RPS5, RAC1, RPP9, CAR1, RPP7, RPW2, RPP1, RPP10, RPP14, RPP5, RPP4, RPS2, RPW6, HRT, RPS4, RPP8, RPP21, RPP22, RPP23, RPP24 and TTR1. This method was therefore not only successful in the identification of sequences located within gene clusters that are involved in disease resistance, but could also contribute to the cloning of disease resistance genes from Arabidopsis.

Amino Acid Sequence↗

PubMed2001

A recessive mutation in the Arabidopsis SSI2 gene confers SA- and NPR1-independent expression of PR genes and resistance against bacterial and oomycete pathogens.

The Arabidopsis thaliana NPR1 gene is required for salicylic acid (SA)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance. However, loss-of-function mutations in NPR1 do not confer complete loss of PR gene expression or disease resistance. Thus these responses also can be activated via an NPR1-independent pathway that currently remain to be elucidated. The ssi2-1 mutant, identified in a genetic screen for suppressors of npr1-5, affects signaling through the NPR1-independent defense pathway(s). In comparison with the wild-type (SSI2 NPR1) plants and the npr1-5 mutant (SSI2 npr1-5), the ssi2-1 npr1-5 double mutant and the ssi2-1 NPR1 single mutant constitutively express PR genes [PR-1, BGL2 (PR-2) and PR-5]; accumulate elevated levels of SA; spontaneously develop lesions; and possess enhanced resistance to a virulent strain of Peronospora parasitica. The ssi2-1 mutation also confers enhanced resistance to Pseudomonas syringae pv. tomato (Pst); however, this is accomplished primarily via an NPR1-dependent pathway. Analysis of ssi2-1 NPR1 nahG and ssi2-1 npr1-5 nahG plants revealed that elevated SA levels were not essential for the ssi2-1-conferred phenotypes. However, expression of the nahG transgene did reduce the intensity of some ssi2-1-conferred phenotypes, including PR-1 expression, and disease resistance. Based on these results, SSI2 or an SSI2-generated signal appears to modulate signaling of an SA-dependent, NPR1-independent defense pathway, or an SA- and NPR1-independent defense pathway.

Arabidopsis↗

PubMed2002

Constitutive expression of a celery mannitol dehydrogenase in tobacco enhances resistance to the mannitol-secreting fungal pathogen Alternaria alternata.

Our previous observation that host plant extracts induce production and secretion of mannitol in the tobacco pathogen Alternaria alternata suggested that, like their animal counterparts, plant pathogenic fungi might produce the reactive oxygen quencher mannitol as a means of suppressing reactive oxygen-mediated plant defenses. The concurrent discovery that pathogen attack induced mannitol dehydrogenase (MTD) expression in the non-mannitol-containing host tobacco suggested that plants, unlike animals, might be able to counter this fungal suppressive mechanism by catabolizing mannitol of fungal origin. To test this hypothesis, transgenic tobacco plants constitutively expressing a celery Mtd cDNA were produced and evaluated for potential changes in resistance to both mannitol- and non-mannitol-secreting pathogens. Constitutive expression of the MTD transgene was found to confer significantly enhanced resistance to A. alternata, but not to the non-mannitol-secreting fungal pathogen Cercospora nicotianae. These results are consistent with the hypothesis that MTD plays a role in resistance to mannitol-secreting fungal plant pathogens.

Alternaria↗

PubMed2003

Silica deposition by a strongly cationic proline-rich protein from systemically resistant cucumber plants.

Infection of one leaf of cucumber (Cucumis sativa) plants can render other leaves resistant to various pathogens. This so-called systemic acquired resistance (SAR) can be functionally mimicked by certain chemicals. All these treatments enhanced expression of a gene encoding a novel proline-rich protein (PRP1) which has C-terminal repetitive sequences containing an unusually high amount of lysine and arginine residues. Antibodies raised against a synthetic peptide derived from four of the repetitive sequences cross-reacted mainly with a cell wall polypeptide of an apparent MW of 8 kDa. The protein accumulated upon SAR induction, though it does not appear to take part in oxidative protein cross-linking, at least in the hypocotyl tissue. The synthetic peptide derived from the repetitive sequences was able to polymerize orthosilicic acid to insoluble silica, a property not resulting directly from the primary protein sequence, but rather from the high positive charge density. Our results suggest that during induction of SAR, the synthesis of a strongly cationic PRP prepares the cell walls for enhanced silica deposition which is known to participate in cell wall reinforcement, localized at the site of attempted penetration of fungi into epidermal cells. Constitutive accumulation of related PRPs may function in silica deposition during certain developmental stages, a process important for various physiological functions of green plants.

Amino Acid Sequence↗

PubMed2004

Root enzyme activities associated with resistance to Heterodera avenae conferred by gene Cre7 in a wheat/Aegilops triuncialis introgression line.

The effect of Cereal cyst nematode (Heterodera avenae) infection on the expression of putative root defence-related enzymes, peroxidase (PER), esterase (EST) and superoxide dismutase (SOD), was studied in roots of a wheat/Aegilops triuncialis introgression line TR-3531 carrying the Cre7 resistance gene. We analysed detoxificant isozyme changes within roots of the resistant line and their susceptible parent (H-10-15) as a control, during the early interaction with the pathotype Ha71 of H. avenae. Isoelectrofocusing (IEF) isozyme analysis, four and seven days after infection, revealed that PER, EST and SOD activities increased in the resistant line TR-3531 in comparison with the susceptible control. Moreover, four and seven days after infection, the TR-3531 line showed the expression of new PER isozymes, with pIs of 9.7, 9.0, 8.5, 6.5 and 5.0, and an increased activity of some constitutive isoforms. The intensity of some EST and SOD constitutive bands increased in the resistant line after infection. However, no new isoforms were detected for EST and SOD systems. Nematode-induced enzyme activity was minor (PER) or did not occur (EST and SOD) in the compatible interaction with H-10-15. The enhanced peroxidase and esterase activities may play a role in the lignification of cell walls, which assists in the resistance to penetration by the nematode.

Animals↗

PubMed2005

Soybean phytophthora resistance gene Rps8 maps closely to the Rps3 region.

Root and stem rot is one of the major diseases of soybean. It is caused by the oomycete pathogen Phytophthora sojae. A series of resistance genes (Rps) have been providing soybean with reasonable protection against this pathogen. Among these genes, Rps8, which confers resistance to most P. sojae isolates, recently has been mapped. However, the most closely linked molecular marker was mapped at about 10 cM from Rps8. In this investigation, we attempted to develop a high-density genetic map of the Rps8 region and identify closely linked SSR markers for marker-assisted selection of this invaluable gene. Bulk segregant analysis was conducted for the identification of SSR markers that are tightly linked to Rps8. Polymorphic SSR markers selected from the Rps8 region failed to show cosegregation with Phytophthora resistance. Subsequently, bulk segregant analysis of the whole soybean genome and mapping experiments revealed that the Rps8 gene maps closely to the disease resistance gene-rich Rps3 region.

Breeding↗

PubMed2003

Induction of resistance and expression of defense-related genes in tobacco leaves infiltrated with Ralstonia solanacearum.

Ralstonia solanacearum 8107 (8107) is non-pathogenic to tobacco and elicits the hypersensitive response (HR). In Nicotiana tabacum cv. Samsun NN leaves infiltrated with 8107, acquired resistance to challenging tobacco mosaic virus (TMV) was induced 2-6 d after 8107-infiltration. hsr203J and hin1 genes were expressed only in the 8107-infiltrated area. On the other hand, the expression of PR-1a and PR-1b genes was not detected in the 8107-infiltrated area, but in areas other than that developing the HR. Expression of these PR-1 genes was regulated simultaneously and the kinetics of the expression was dependent on the distance from the infiltration area. Therefore, diffusible signal(s) might be produced in HR-causing cells and transmitted to peripheral cells resulting in expression of PR genes. In NahG10 tobacco infiltrated with 8107, the HR was induced but resistance to TMV was not. Analysis using NahG10 tobacco also showed that the salicylic acid (SA)-dependent signal regulated the expression of hsr203J and PR-1a, but not that of hin1 and PR-1b. These results suggest that resistance of tobacco to 8107 is SA-independent and involves a quite different mechanism from acquired resistance to TMV induced by 8107-infiltration which is SA-dependent.

Amino Acid Sequence↗

PubMed2005

Fusarium oxysporum evades I-3-mediated resistance without altering the matching avirulence gene.

I-3-Mediated resistance of tomato against Fusarium wilt disease caused by Fusarium oxysporum f. sp. lycopersici depends on Six1, a protein that is secreted by the fungus during colonization of the xylem. Among natural isolates of F. oxysporum f. sp. lycopersici are several that are virulent on a tomato line carrying only the I-3 resistance gene. However, evasion of I-3-mediated resistance by these isolates is not correlated with mutation of the SIX1 gene. Moreover, the SIX1 gene of an I-3-virulent isolate was shown to be fully functional in that i) the gene product is secreted in xylem sap, ii) deletion leads to a further increase in virulence on the I-3 line as well as reduced virulence on susceptible lines, and iii) the gene confers full avirulence on the I-3 line when transferred to another genetic background. Remarkably, all I-3-virulent isolates were of race 1, suggesting a link between the presence of AVR1 and evasion of I-3-mediated resistance.

Fungal Proteins↗