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PubMed2002

Structural analysis of the maize rp1 complex reveals numerous sites and unexpected mechanisms of local rearrangement.

Rp1 is a complex disease resistance locus in maize that is exceptional in both allelic variability and meiotic instability. Genomic sequence analysis of three maize BACs from the Rp1 region of the B73 inbred line revealed 4 Rp1 homologs and 18 other gene-homologous sequences, of which at least 16 are truncated. Thirteen of the truncated genes are found in three clusters, suggesting that they arose from multiple illegitimate break repairs at the same sites or from complex repairs of each of these sites with multiple unlinked DNA templates. A 43-kb region that contains an Rp1 homolog, six truncated genes, and three Opie retrotransposons was found to be duplicated in this region. This duplication is relatively recent, occurring after the insertion of the three Opie elements. The breakpoints of the duplication are outside of any genes or identified repeat sequence, suggesting a duplication mechanism that did not involve unequal recombination. A physical map and partial sequencing of the Rp1 complex indicate the presence of 15 Rp1 homologs in regions of approximately 250 and 300 kb in the B73 inbred line. Comparison of fully sequenced Rp1-homologous sequences in the region demonstrates a history of unequal recombination and diversifying selection within the Leu-rich repeat 2 region, resulting in chimeric gene structures.

Chromosome Mapping↗
PubMed2003

Disease resistance and abiotic stress tolerance in rice are inversely modulated by an abscisic acid-inducible mitogen-activated protein kinase.

Mitogen-activated protein kinase (MAPK) cascades play an important role in mediating stress responses in eukaryotic organisms. However, little is known about the role of MAPKs in modulating the interaction of defense pathways activated by biotic and abiotic factors. In this study, we have isolated and functionally characterized a stress-responsive MAPK gene (OsMAPK5) from rice. OsMAPK5 is a single-copy gene but can generate at least two differentially spliced transcripts. The OsMAPK5 gene, its protein, and kinase activity were inducible by abscisic acid as well as various biotic (pathogen infection) and abiotic (wounding, drought, salt, and cold) stresses. To determine its biological function, we generated and analyzed transgenic rice plants with overexpression (using the 35S promoter of Cauliflower mosaic virus) or suppression (using double-stranded RNA interference [dsRNAi]) of OsMAPK5. Interestingly, suppression of OsMAPK5 expression and its kinase activity resulted in the constitutive expression of pathogenesis-related (PR) genes such as PR1 and PR10 in the dsRNAi transgenic plants and significantly enhanced resistance to fungal (Magnaporthe grisea) and bacterial (Burkholderia glumae) pathogens. However, these same dsRNAi lines had significant reductions in drought, salt, and cold tolerance. By contrast, overexpression lines exhibited increased OsMAPK5 kinase activity and increased tolerance to drought, salt, and cold stresses. These results strongly suggest that OsMAPK5 can positively regulate drought, salt, and cold tolerance and negatively modulate PR gene expression and broad-spectrum disease resistance.

Abscisic Acid↗
PubMed1997

The Arabidopsis downy mildew resistance gene RPP5 shares similarity to the toll and interleukin-1 receptors with N and L6.

Plant disease resistance genes operate at the earliest steps of pathogen perception. The Arabidopsis RPP5 gene specifying resistance to the downy mildew pathogen Peronospora parasitica was positionally cloned. It encodes a protein that possesses a putative nucleotide binding site and leucine-rich repeats, and its product exhibits striking structural similarity to the plant resistance gene products N and L6. Like N and L6, the RPP5 N-terminal domain resembles the cytoplasmic domains of the Drosophila Toll and mammalian interleukin-1 transmembrane receptors. In contrast to N and L6, which produce predicted truncated products by alternative splicing, RPP5 appears to express only a single transcript corresponding to the full-length protein. However, a truncated form structurally similar to those of N and L6 is encoded by one or more other members of the RPP5 gene family that are tightly clustered on chromosome 4. The organization of repeated units within the leucine-rich repeats encoded by the wild-type RPP5 gene and an RPP5 mutant allele provides molecular evidence for the heightened capacity of this domain to evolve novel configurations and potentially new disease resistance specificities.

Alleles↗
PubMed2002

Regulatory role of SGT1 in early R gene-mediated plant defenses.

Animal SGT1 is a component of Skp1-Cullin-F-box protein (SCF) ubiquitin ligases that target regulatory proteins for degradation. Mutations in one (SGT1b) of two highly homologous Arabidopsis SGT1 genes disable early plant defenses conferred by multiple resistance (R) genes. Loss of SGT1b function in resistance is not compensated for by SGT1a. R genes differ in their requirements for SGT1b and a second resistance signaling gene, RAR1, that was previously implicated as an SGT1 interactor. Moreover, SGT1b and RAR1 contribute additively to RPP5-mediated pathogen recognition. These data imply both operationally distinct and cooperative functions of SGT1 and RAR1 in plant disease resistance.

Amino Acid Motifs↗
PubMed2005

[Cloning and analysis of a new NBS-LRR resistance gene family in rice].

Sequence-based gene isolation has been a practical approach for plant resistance gene cloning. In this study, RS13, a cloned rice sequence with the NBS (nucleotide-binding site) domain of resistance genes, was used as a probe to screen a bacterial artificial chromosome (BAC) library of rice variety IR64,and four positive clones were obtained. Of them the clone 14E19 covered the other three clones and was sequenced through a shotgun approach. The whole sequence of the insert fragment of 14E19 was assembled into approximately 73 kb in length. Genes on the whole assembled sequence were predicted,and four genes encoding NBS and LRR (leucine-rich repeat) domains were found, named as NL-A, B, C and D respectively. For further analysis, another longer BAC clone,106P13, covering 14E19 on the same chromosome position was identified from a BAC library of IRBB56 which had the same genome background with IR64. Ten NL-homologous copies were discovered on the sequence of the BAC clone 106P13, and four copies were identical with those on 14E19. The similar homologous sequences were also found in the genomic sequences of Nipponbare,93-11 and Guangluai4. However, NL sequences were less homologous with the known NBS-LRR resistance genes. This result indicated that NL was a new NBS-LRR gene family and was composed of ten members at least. RT-PCR and cDNA screening displayed that NL-B expressed in a bacterial blight-resistant rice variety IRBB4, indicating the gene was possibly involved in resistance reactions.

Amino Acid Sequence↗
PubMed1984

Monoclonal antibodies in plant virology.

Monoclonal antibodies specific for more than 20 plant viruses have been obtained. They have been found very useful for large-scale diagnosis of viral diseases and for elucidating the antigenic structure of plant viruses at the molecular level. The advantages and limitations of these new reagents and some of their applications in plant virus serology are discussed.

Antibodies, Monoclonal↗
PubMed2004

Characterization of an NBS-LRR resistance gene homologue from soybean.

Conserved motifs such as the nucleotide-binding site (NBS) were found in many characterized plant disease resistance genes. Based on the NBS domain, resistance gene analogs have been isolated in our previous study and were used as probes to screen a soybean (Glycine max) cDNA library. A full-length cDNA, KR4, was isolated by screening the library and rapid amplification of cDNA ends method. Sequence analysis revealed that the cDNA was 3818 bp in length and the open reading frame coded for a polypeptide of 1211 amino acids with an NBS and five leucine-rich repeats domains, which were identified by Pfam protein analysis. Sequence alignment showed that KR4 was similar to 12 protein of tomato. Southern analysis indicated that the KR4 gene had low copies in soybean genome and it was mapped on the molecular linkage group E. Its expression was also investigated and it was found that KR4 was induced by exogenous salicylic acid and responded upon infection of soybean mosaic virus strain N3.

Amino Acid Sequence↗
PubMed2002

Over-expression of TGA5, which encodes a bZIP transcription factor that interacts with NIM1/NPR1, confers SAR-independent resistance in Arabidopsis thaliana to Peronospora parasitica.

The Arabidopsis thaliana NIM1/NPR1 gene product is required for induction of systemic acquired resistance (SAR) by pathogens, salicylic acid (SA) or synthetic SA analogs. We identified, in a yeast two-hybrid screen, two NIM1/NPR1 interacting proteins, TGA2 and TGA5, which belong to the basic region, leucine zipper (bZIP) family of transcription factors. Both TGA2 and TGA5 strongly interact with NIM1/NPR1 in yeast and in vitro, and recognize the as-1 cis element found within the promoter of several pathogenesis-related genes, such as PR-1. To determine the role TGA2 and TGA5 may play in NIM1/NPR1-mediated disease resistance, we introduced sense and antisense versions of both genes into transgenic Arabidopsis plants. Characterization of TGA2 transgenic plants revealed that inhibition or overexpression of TGA2 does not significantly affect PR-1 expression or induction of SAR after pathogen infection or INA treatment. Surprisingly, all TGA5-antisense transgenic plants produced showed increased accumulation of TGA5 transcripts compared with untransformed control plants, while the TGA5-sense lines showed no significant increase in TGA5 mRNA levels. Interestingly, the high level of TGA5 mRNA in the antisense lines was accompanied by significant resistance to a highly virulent isolate of the oomycete pathogen Peronospora parasitica. Further, resistance was not coupled to accumulation of products from the SAR-linked PR-1 gene following inoculation with P. parasitica or treatment with INA, indicating that these plants express a robust, PR-1-independent resistance mechanism. Resistance was retained when a TGA5-accumulating line was combined genetically with a nim1-1 mutation or nahG (salicylate hydroxylase) transgene, indicating that resistance in these plants is due to an SA and SAR-independent mechanism.

Antisense Elements (Genetics)↗
PubMed2002

A gain-of-function mutation in an Arabidopsis Toll Interleukin1 receptor-nucleotide binding site-leucine-rich repeat type R gene triggers defense responses and results in enhanced disease resistance.

In a screen for suppressors of npr1-5-based salicylic acid (SA) insensitivity, we isolated a semidominant gain-of-function mutation, designated ssi4, that confers constitutive expression of several PR (pathogenesis-related) genes, induces SA accumulation, triggers programmed cell death, and enhances resistance to bacterial and oomycete pathogens. Through map-based cloning, ssi4 was identified and found to encode a putative protein belonging to the TIR-NBS-LRR (Toll Interleukin1 Receptor-Nucleotide Binding Site-Leu-Rich Repeat) class of R (resistance) proteins. Comparison between ssi4 and the corresponding wild-type sequence revealed a single amino acid substitution in the NBS. Epistasis analysis indicated that SA and EDS1 are required for ssi4-induced PR-1 expression and enhanced disease resistance; they also are required for the increased accumulation of SSI4 and EDS1 transcripts detected in the ssi4 mutant. Although high levels of ssi4 transcripts correlate with the appearance of the mutant phenotype, overexpression of the wild-type SSI4 gene failed to induce stunting, spontaneous lesion formation, or increased PR-1 expression associated with the ssi4 mutation. Thus, the ssi4 phenotype does not appear to be caused by overexpression of this R gene; rather, we propose that the NBS substitution generates a constitutively activated R protein. Furthermore, because SA treatment induced the expression of SSI4 and the closely related TIR-NBS-LRR genes RPP1 and RPS4 but had little effect on the expression of the coiled-coil NBS-LRR genes RPM1 and RPS2, we suggest that SA not only functions as a critical signal for downstream resistance events but also upregulates the expression of certain R genes.

Amino Acid Sequence↗
PubMed2003

The tomato suppressor of prosystemin-mediated responses2 gene encodes a fatty acid desaturase required for the biosynthesis of jasmonic acid and the production of a systemic wound signal for defense gene expression.

Genetic analysis of the wound response pathway in tomato indicates that systemin and its precursor protein, prosystemin, are upstream components of a defensive signaling cascade that involves the synthesis and subsequent action of the octadecatrienoic acid (18:3)-derived plant hormone jasmonic acid (JA). The suppressor of prosystemin-mediated responses2 (spr2) mutation, which was isolated previously as a suppressor of (pro)systemin-mediated signaling, impairs wound-induced JA biosynthesis and the production of a long-distance signal for the expression of defensive Proteinase inhibitor genes. Using a map-based cloning approach, we demonstrate here that Spr2 encodes a chloroplast fatty acid desaturase involved in JA biosynthesis. Loss of Spr2 function reduced the 18:3 content of leaves to <10% of wild-type levels, abolished the accumulation of hexadecatrienoic acid, and caused a corresponding increase in the level of dienoic fatty acids. The effect of spr2 on the fatty acyl content of various classes of glycerolipids indicated that the Spr2 gene product catalyzes most, if not all, omega3 fatty acid desaturation within the "prokaryotic pathway" for lipid synthesis in tomato leaves. Despite the reduced levels of trienoic fatty acids, spr2 plants exhibited normal growth, development, and reproduction. However, the mutant was compromised in defense against attack by tobacco hornworm larvae. These results indicate that jasmonate synthesis from chloroplast pools of 18:3 is required for wound- and systemin-induced defense responses and support a role for systemin in the production of a transmissible signal that is derived from the octadecanoid pathway.

Animals↗
PubMed2005

A putative nucleoporin 96 Is required for both basal defense and constitutive resistance responses mediated by suppressor of npr1-1,constitutive 1.

The Arabidopsis thaliana suppressor of npr1-1, constitutive 1 (snc1) mutant contains a gain-of-function mutation in a Toll Interleukin1 receptor-nucleotide binding-Leu-rich repeat-type resistance gene (R-gene), which leads to constitutive activation of disease resistance response against pathogens. In a screen for suppressors of snc1, a recessive mutation, designated mos3 (for modifier of snc1,3), was found to suppress the constitutive pathogenesis-related gene expression and resistance to virulent Pseudomonas syringae maculicola ES4326 and Peronospora parasitica Noco2 in snc1. In addition, mos3 is also compromised in resistance mediated by Resistance to Peronospora parasitica 4 (RPP4), Resistance to Pseudomonas syringae pv maculicola (RPM1), and Resistance to Pseudomonas syringae 4 (RPS4). Single mutant mos3 plants exhibited enhanced disease susceptibility to P. s. pv maculicola ES4326, suggesting that MOS3 is required for basal resistance to pathogens as well. mos3-1 was identified by map-based cloning, and it encodes a protein with high sequence similarity to human nucleoporin 96. Localization of the MOS3-green fluorescent protein fusion to the nuclear envelope further indicates that MOS3 may encode a nucleoporin, suggesting that nuclear and cytoplasmic trafficking plays an important role in both R-gene-mediated and basal disease resistance.

Active Transport, Cell Nucleus↗
PubMed2005

[Identification of genes related to resistance to Magnaporthe grisea using differential display technique in rice].

Rice blast caused by Magnaporthe grisea is one of the most serious constraints on high productivity. Understanding the mechanism of the infection of Magnaporthe grisea and the change of gene expression after infection is useful to control blast disease in rice. This work presents the isolation of differentially expressed cDNA fragments from rice leaf induced by the inoculum suspension of Magnaporthe grisea using mRNA differential display technique. Total 87 differential expressed cDNA fragments were recoveried and reamplified. The dot-blotting results showed that 6 fragments of 81 were confirmed to be the expression induced by Magnaporthe grisea inoculum. Those fragments were then cloned into vectors for sequencing. Sequence analysis through Internet Blast searching showed that 3 fragments were novel gene fragments. One was homologous with a putative malate synthase gene on rice chromosome 4 with 78% identities of amino acid; one was highly homologous (75% identity) with rice RPR1 gene on chromosome 11, which has a conservative structure of NBS-LRR domain and may be related to signal transduction of rice defense reaction;another one was homologous with a putative thioredoxin gene on rice chromosome 6 with the identity of 72%.

Base Sequence↗
PubMed1999

Protection of mice against challenge with foot and mouth disease virus (FMDV) by immunization with foliar extracts from plants infected with recombinant tobacco mosaic virus expressing the FMDV structural protein VP1.

A tobacco mosaic virus (TMV)-based vector has been used to express in plants the complete open reading frame coding for VP1, the major immunogenic protein of foot and mouth disease virus (FMDV). In vitro RNA transcripts were inoculated into Nicotiana benthamiana plants and detectable amounts of recombinant VP1 were identified by Western blot as soon as 4 days postinfection. Foliar extracts prepared from infected leaves were injected intraperitoneally into mice and all of the immunized animals developed a specific antibody response to both the complete virus particle and the major immunogenic region as determined by ELISA and Western blot analysis. Most importantly, all immunized mice developed a protective immune response against experimental challenge with virulent FMDV. To our knowledge, this is the first report showing the expression of a complete open reading frame of an antigenic foreign protein in plants, using a recombinant plant virus, in sufficient quantity to permit use of the crude plant extract as an experimental immunogen to protect animals against virus challenge.

Animals↗
PubMedDate not supplied

Specific immune response to Parietaria judaica plant profilin: a low T cell proliferative response supports high IgE and skin prick test.

BACKGROUND: allergic disease caused by Parietaria judaica (Pj) has been widely documented in Mediterranean area. Profilins have been identified as widely distributed allergenic proteins. The role of Pj profilin in specific immune response in Pj-sensitized patients is unknown. METHODS: skin prick test and determination of specific and total IgE levels in serum were performed in all patients (n = 28) and non-allergic controls (n = 18). Peripheral blood mononuclear cells (PBMC) were isolated from both groups and stimulated with crude extract or highly purified Pj profilin. The production of type I and type II cytokines was determined by specific and polyclonal stimuli in patients and controls. T-cell lines specific to Pj profilin were established and cross-reactivity with another highly purified profilin from Phleum pratense (Phl p) was evaluated. RESULTS: Pj profilin-sensitized patients showed a small but significantly increased in T-cell proliferative response to this profilin compared with non-atopic controls. The production of interleukin (IL)-4 and interferon (IFN)-γ in response to the specific stimulus was undetectable. However, the production of IL-4 in response to a polyclonal stimulus [phytohemagglutinin (PHA)] was significantly higher in atopic patients than in controls. The T-cell response did not correlate with the magnitude of response to skin prick tests with Pj profilin or with Pj-specific serum IgE levels. In addition, the production of IL-4 in response to a polyclonal stimulus (PHA) did not correlate with the individual skin prick tests to Pj profilin or with Pj-specific IgE levels in serum. The T-cell lines tested showed no cross-reactivity with Phl p profilin. CONCLUSIONS: our results suggest that Pj profilin is partly responsible for the T-cell-mediated response in patients allergic to Pj. The high skin reactivity to Pj profilin is these patients was accompanied by a small increase in the T-cell response to this profilin. The response was highly specific since Pj profilin specific T-cell lines showed no cross-reactivity with a highly homologous profilin from Phl p. The lack of correlation between the proliferative T-cell response and polyclonal IL-4 production with allergen-specific serum IgE and skin reactivity probably indicates that some of the responding T-cells may be involved in immune reactions other than those supporting IgE production.

Adult↗
PubMed1998

Identification and Ds-tagged isolation of a new gene at the Cf-4 locus of tomato involved in disease resistance to Cladosporium fulvum race 5.

Leaf mould disease in tomato is caused by the biotrophic fungus Cladosporium fulvum. An Ac/Ds targeted transposon tagging strategy was used to isolate the gene conferring resistance to race 5 of C. fulvum, a strain expressing the avirulence gene Avr4. An infection assay of 2-week-old seedlings yielded five susceptible mutants, of which two had a Ds element integrated in the same gene at different positions. This gene, member of a gene family, showed high sequence homology to the C. fulvum resistance genes Cf-9 and Cf-2. The gene is predicted to encode an extracellular transmembrane protein containing a divided domain of 25 leucine-rich repeats. Three mutants exhibited a genomic deletion covering most of the Lycopersicon hirsutum introgressed segment, including the Cf-4 locus. Southern blot analysis revealed that this deletion includes the tagged gene and five homologous sequences. To test whether the tagged gene confers resistance to C. fulvum via Avr4 recognition, the Avr4 gene was expressed in planta. Surprisingly, expression of the Avr4 gene still triggered a specific necrotic response in the transposon-tagged plants, indicating that the tagged resistance gene is not, or is not the only gene, involved in Avr4 recognition. Mutants harbouring the genomic deletion did not show this Avr4-specific response. The deleted segment apparently contains, in addition to the tagged gene, one or more other genes, which play a role in the Avr4 responses. The tagged gene is present at the Cf-4 locus, but it does not necessarily recognize Avr4 and is therefore designated Cf-4A.

Amino Acid Sequence↗
PubMed2000

Flagellin from an incompatible strain of Pseudomonas avenae induces a resistance response in cultured rice cells.

The host range of Pseudomonas avenae is wide among monocotyledonous plants, but individual strains can infect only one or a few host species. The resistance response of rice cells to pathogens has been previously shown to be induced by a rice-incompatible strain, N1141, but not by a rice-compatible strain, H8301. To clarify the molecular mechanism of the host specificity in P. avenae, a strain-specific antibody that was raised against N1141 cells and then absorbed with H8301 cells was prepared. When a cell extract of strain N1141 was separated by SDS-polyacrylamide gel electrophoresis and immunostained with the N1141 strain-specific antibody, only a flagellin protein was detected. Purified N1141 flagellin induced the hypersensitive cell death in cultured rice cells within 6 h of treatment, whereas the H8301 flagellin did not. The hypersensitive cell death could be blocked by pretreatment with anti-N1141 flagellin antibody. Furthermore, a flagellin-deficient N1141 strain lost not only the induction ability of hypersensitive cell death but also the expression ability of the EL2 gene, which is thought to be one of the defense-related genes. These results demonstrated that the resistance response in cultured rice cells is induced by the flagellin existing in the incompatible strain of P. avenae but not in the flagellin of the compatible strain.

Amino Acid Sequence↗