Mutations in PMR5 result in powdery mildew resistance and altered cell wall composition

Powdery mildews and other obligate biotrophic pathogens are highly adapted to their hosts and often show limited host ranges. One facet of such host specialization is likely to be penetration of the host cell wall, a major barrier to infection. A mutation in the pmr5 gene rendered Arabidopsis resistant to the powdery mildew species Erysiphe cichoracearum and Erysiphe orontii, but not to the unrelated pathogens Pseudomonas syringae or Peronospora parasitica. PMR5 belongs to a large family of plant-specific genes of unknown function. pmr5-mediated resistance did not require signaling through either the salicylic acid or jasmonic acid/ethylene defense pathways, suggesting resistance in this mutant may be due either to the loss of a susceptibility factor or to the activation of a novel form of defense. Based on Fourier transform infrared analysis, the pmr5 cell walls were enriched in pectin and exhibited a reduced degree of pectin modification relative to wild-type cell walls. In addition, the mutant had smaller cells, suggesting a defect in cell expansion. A double mutant with pmr6 (defective in a glycosylphosphatidylinositol-anchored pectate lyase-like gene) exhibited a strong increase in total uronic acid content and a more severe reduction in size, relative to the single mutants, suggesting that the two genes affect pectin composition, either directly or indirectly, via different mechanisms. These two mutants highlight the importance of the host cell wall in plant-microbe interactions.     

Regulation of plant defense responses in Arabidopsis by EDR2, a PH and START domain-containing protein

We have identified an Arabidopsis mutant that displays enhanced disease resistance (edr2) to the biotrophic powdery mildew pathogen Erysiphe cichoracearum. Inhibition of fungal growth on edr2 mutant leaves occurred at a late stage of the infection process and coincided with formation of necrotic lesions approximately 5 days after inoculation. Double-mutant analysis revealed that edr2-mediated resistance is suppressed by mutations that inhibit salicylic acid (SA)-induced defense signaling, including npr1, pad4 and sid2, demonstrating that edr2-mediated disease resistance is dependent on SA. However, edr2 showed normal responses to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000. EDR2 appears to be constitutively transcribed in all tissues and organs and encodes a novel protein, consisting of a putative pleckstrin homology (PH) domain and a steroidogenic acute regulatory protein-related lipid-transfer (START) domain, and contains an N-terminal mitochondrial targeting sequence. The PH and START domains are implicated in lipid binding, suggesting that EDR2 may provide a link between lipid signaling and activation of programmed cell death mediated by mitochondria.     

Loss of actin cytoskeletal function and EDS1 activity,in combination,severely compromises non-host resistance in Arabidopsis against wheat powdery mildew

Plant immunity against the majority of the microbial pathogens is conveyed by a phenomenon known as non-host resistance (NHR). This defence mechanism affords durable protection to plant species against given species of phytopathogens. We investigated the genetic basis of NHR in Arabidopsis against the wheat powdery mildew fungus Blumeria graminis f. sp. tritici (Bgt). Both primary and appressorial germ tubes were produced from individual Bgt conidia on the surface of the Arabidopsis leaves. Attempted infection occasionally resulted in successful penetration, which led to the development of an abnormal unilateral haustorium. Inoculation of a series of Arabidopsis defence-related mutants with Bgt resulted in the attenuation of reactive oxygen intermediate (ROI) production and salicylic acid (SA)-dependent defence gene expression in eds1, pad4 and nahG plants, which are known to be defective in some aspects of host resistance. Furthermore, Bgt often developed bilateral haustoria in the mutant Arabidopsis lines that closely resembled those formed in wheat. A similar decrease in NHR was observed following treatment of the wild-type Arabidopsis plants with cytochalasin E, an inhibitor of actin microfilament polymerisation. In eds1 mutants, inhibition of actin polymerisation severely compromised NHR in Arabidopsis against Bgt. This permitted completion of the Bgt infection cycle on these plants. Therefore, actin cytoskeletal function and EDS1 activity, in combination, are major contributors to NHR in Arabidopsis against wheat powdery mildew.     

Structure,expression and localization of a germin-like protein in barley (Hordeum vulgare L.) that is insolubilized in stressed leaves

The primary leaves of young barley seedlings contain two major, extracellular, acid-soluble proteins of ca. 22 and 23 kDa apparent molecular mass. These proteins disappeared from the intercellular washing fluid upon stress treatments that enhanced H₂O₂ levels and that induced resistance to subsequent challenge by the powdery mildew fungus Erysiphe graminis f. sp. hordei. A partial peptide sequence of the 22 kDa protein was determined, and a cDNA clone was isolated. The 22 kDa protein belongs the the group of germin-like proteins (GLPs) and was designated HvGLP1. Despite its similarity to germin, i.e. oxalate oxidase, no oxalate oxidase activity of HvGLP1 could be detected. The RNA and soluble protein of HvGLP1 was highly abundant in young leaves, less abundant in older leaves and absent in roots. HvGLP1 RNA oscillated with a circadian rhythm, the minimum and maximum of RNA abundance being at the end of the dark and light periods, respectively. Heat and H₂O₂ treatment as well as pathogen infection caused disappearance of HvGLP1 protein from the fraction of soluble proteins of the intercellular space. HvGLP1 protein could be re-solubilized from cell walls of heat- or H₂O₂-treated leaves by boiling in SDS suggesting non-covalent cross linking. Although a physiological role of HvGLP1 insolubilization is still open, the protein may serve as marker for oxidative stress in cereals.     

A mutation in the GTP hydrolysis site of Arabidopsis dynamin-related protein 1E confers enhanced cell death in response to powdery mildew infection

We screened for mutants of Arabidopsis thaliana that displayed enhanced disease resistance to the powdery mildew pathogen Erysiphe cichoracearum and identified the edr3 mutant, which formed large gray lesions upon infection with E. cichoracearum and supported very little sporulation. The edr3-mediated disease resistance and cell death phenotypes were dependent on salicylic acid signaling, but independent of ethylene and jasmonic acid signaling. In addition, edr3 plants displayed enhanced susceptibility to the necrotrophic fungal pathogen Botrytis cinerea, but showed normal responses to virulent and avirulent strains of Pseudomonas syringae pv. tomato. The EDR3 gene was isolated by positional cloning and found to encode Arabidopsis dynamin-related protein 1E (DRP1E). The edr3 mutation caused an amino acid substitution in the GTPase domain of DRP1E (proline 77 to leucine) that is predicted to block GTP hydrolysis, but not GTP binding. A T-DNA insertion allele in DRP1E did not cause powdery mildew-induced lesions, suggesting that this phenotype is caused by DRP1E being locked in the GTP-bound state, rather than by a loss of DRP1E activity. Analysis of DRP1E-green fluorescent protein fusion proteins revealed that DRP1E is at least partially localized to mitochondria. These observations suggest a mechanistic link between salicylic acid signaling, mitochondria and programmed cell death in plants.     

Down-regulation of an Auxin Response Factor in the tomato induces modification of fine pectin structure and tissue architecture

It has previously been shown that down-regulation of an auxin response factor gene (DR12) results in pleiotropic phenotypes including enhanced fruit firmness in antisense transgenic tomato (AS-DR12). To uncover the nature of the ripening-associated modifications affecting fruit texture, comparative analyses were performed of pectin composition and structure in cell wall pericarp tissue of wild-type and AS-DR12 fruit at mature green (MG) and red-ripe (RR) stages. Throughout ripening, pectin showed a decrease in methyl esterification and in the content of galactan side chains in both genotypes. At mature green stage, pectin content in methyl ester groups was slightly higher in AS-DR12 fruit than in wild type, but this ratio was reversed at the red-ripe stage. The amount of water- and oxalate-soluble pectins increased at the red-ripe stage in the wild type, but decreased in AS-DR12. The distribution of methyl ester groups on the homogalaturonan backbone differed between the two genotypes. There was no evidence of more calcium cross-linked homogalacturan involved in cell-to-cell adhesion in AS-DR12 compared with wild-type fruit. Furthermore, the outer pericarp contains higher proportion of small cells in AS-DR12 fruit than in wild type and higher occurrence of (1-->5) alpha-L-arabinan epitope at the RR stage. It is concluded that the increased firmness of transgenic fruit does not result from a major impairment of ripening-related pectin metabolism, but rather involves differences in pectin fine structure associated with changes in tissue architecture.     

Genotypic differences in Al resistance and the role of cell-wall pectin in Al exclusion from the root apex in Fagopyrum tataricum

Background and Aims

Aluminium (Al) toxicity is one of the factors limiting crop production on acid soils. However, genotypic differences exist among plant species or cultivars in response to Al toxicity. This study aims to investigate genotypic differences among eight cultivars of tatary buckwheat (Fagopyrum tataricum) for Al resistance and explore the possible mechanisms of Al resistance.

Methods

Al resistance was evaluated based on relative root elongation (root elongation with Al/root elongation without Al). Root apex Al content, pectin content and exudation of root organic acids were determined and compared.

Key Results

Genotypic differences among the eight cultivars were correlated with exclusion of Al from the root apex. However, there was a lack of correlation between Al exclusion and Al-induced oxalate secretion. Interestingly, cell-wall pectin content of the root apex was generally lower in Al-resistant cultivars than in Al-sensitive cultivars. Although we were unable to establish a significant correlation between Al exclusion and pectin content among the eight cultivars, a strong correlation could be established among six cultivars, in which the pectin content in the most Al-resistant cultivar ‘Chuan’ was significantly lower than that in the most Al-sensitive cultivar ‘Liuku2’. Furthermore, root apex cell-wall pectin methylesterase activity (PME) was similar in ‘Chuan’ and ‘Liuku2’ in the absence of Al, but Al treatment resulted in increased PME activity in ‘Liuku2’ compared with ‘Chuan’. Immunolocalization of pectins also showed that the two cultivars had similar amounts of either low-methyl-ester pectins or high-methyl-ester pectins in the absence of Al, but Al treatment resulted in a more significant increase of low-methyl-ester pectins and decrease of high-methyl-ester pectins in ‘Liuku2’.

Conclusions

Cell-wall pectin content may contribute, at least in part, to differential Al resistance among tatary buckwheat cultivars.     

A common plant cell-wall protein HyPRP1 has dual roles as a positive regulator of cell death and a negative regulator of basal defense against pathogens

Although hybrid proline-rich proteins (HyPRPs) are ubiquitous in plants, little is known about their roles other than as cell-wall structural proteins. We identified the gene HyPRP1 in Capsicum annuum and Nicotiana benthamiana, which encodes a protein containing proline-rich domain and eight-cysteine motif (8CM) that is constitutively expressed in various organs, mostly in the root, but is down-regulated upon inoculation with either incompatible or compatible pathogens. Ectopic expression of HyPRP1 in plants accelerated cell death, showing developmental abnormality with down-regulation of ROS-scavenging genes, and enhanced pathogen susceptibility suppressing expression of defense-related genes. Conversely, silencing of HyPRP1 suppressed pathogen-induced cell death, but enhanced disease resistance, with up-regulation of defense-related genes and inhibition of in planta growth of bacterial pathogens independently of signal molecule-mediated pathways. Furthermore, the secreted 8CM was sufficient for these HyPRP1 functions. Together, our results suggest that a common plant cell-wall structural protein, HyPRP1, performs distinct dual roles in positive regulation of cell death and negative regulation of basal defense against pathogen.     

Predators mediate the effects of a fungal pathogen on prey: an experiment with grasshoppers,wolf spiders,and fungal pathogens

1. Prey interact with multiple kinds of enemies such as predators, parasites, and pathogens. Interactions among enemies can alter prey dynamics but they are often studied separately. 2. During the summers of 2005–2006, we conducted a field experiment to examine interactions among grasshoppers, spider predators, and a lethal fungal pathogen of grasshoppers. Grasshopper nymphs were stocked into field enclosures. Predation was manipulated by adding spiders to enclosures on day 1, day 5, or day 10 of the experiment, or no spiders were added. We monitored grasshopper survival and grasshopper mortality from fungal pathogens for 4 weeks. 3. Fungal pathogens were abundant in 2005 but not in 2006, probably because of favourable weather conditions in 2005. When fungal pathogens were abundant, spider presence reduced grasshopper mortality from fungal pathogens, but only when spiders were present early in the experiment (added on day 1 or day 5). 4. The outcome of predator–prey interactions varied between years, probably as a result of differences in pathogen prevalence. In 2005, spider presence reduced the number of deaths from the pathogen, leading to a slight trend of increased grasshopper density. However, in 2006, when pathogens were not an important source of mortality, spider predation was compensatory.     

Elaborate spatial patterning of cell-wall PME and PMEI at the pollen tube tip involves PMEI endocytosis, and reflects the distribution of esterified and de-esterified pectins

In dicots, pectins are the major structural determinant of the cell wall at the pollen tube tip. Recently, immunological studies revealed that esterified pectins are prevalent at the apex of growing pollen tubes, where the cell wall needs to be expandable. In contrast, lateral regions of the cell wall contain mostly de-esterified pectins, which can be cross-linked to rigid gels by Ca(2+) ions. In pollen tubes, several pectin methylesterases (PMEs), enzymes that de-esterify pectins, are co-expressed with different PME inhibitors (PMEIs). This raises the possibility that interactions between PMEs and PMEIs play a key role in the regulation of cell-wall stability at the pollen tube tip. Our data establish that the PME isoform AtPPME1 (At1g69940) and the PMEI isoform AtPMEI2 (At3g17220), which are both specifically expressed in Arabidopsis pollen, physically interact, and that AtPMEI2 inactivates AtPPME1 in vitro. Furthermore, transient expression in tobacco pollen tubes revealed a growth-promoting activity of AtPMEI2, and a growth-inhibiting effect of AtPPME1. Interestingly, AtPPME1:YFP accumulated to similar levels throughout the cell wall of tobacco pollen tubes, including the tip region, whereas AtPMEI2:YFP was exclusively detected at the apex. In contrast to AtPPME1, AtPMEI2 localized to Brefeldin A-induced compartments, and was found in FYVE-induced endosomal aggregates. Our data strongly suggest that the polarized accumulation of PMEI isoforms at the pollen tube apex, which depends at least in part on local PMEI endocytosis at the flanks of the tip, regulates cell-wall stability by locally inhibiting PME activity.     

The pattern of distribution of pectin, peroxidase and lignin in the middle lamella of secondary xylem fibres in alfalfa (Medicago sativa)

BACKGROUNDS AND AIMS: Information on the micro-distribution of lignin within the middle lamella is only just beginning to emerge. This paper provides evidence of marked heterogeneity in the micro-distribution of lignin, pectin, peroxidase and hydrogen peroxide in the middle lamella of alfalfa (Medicago sativa). METHODS: Specimens from alfalfa stems were collected and processed for transmission electron microscopy. The middle lamella architecture was examined prior to and during lignification, using transmission electron microscopy in combination with pectin- and lignin-specific staining. In addition, immuno-gold labelling of peroxidase and cytochemical localization of hydrogen peroxide (H2O2) were undertaken. KEY RESULTS: Lignin showed inhomogeneity in its distribution in the middle lamella. It was found that the distribution of pectin was irregular and corresponded to the pattern of deposited lignin. Additionally, a similarity in the pattern of the deposited lignin to the pattern of distribution of peroxidase and H2O2 was also observed. CONCLUSIONS: Irregular distribution of pectin in the middle lamella may be related to subsequent inhomegeneity in lignin in this region.     

Genetic dissection of a TIR‐NB‐LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine

The most economically important diseases of grapevine cultivation worldwide are caused by the fungal pathogen powdery mildew (Erysiphe necator syn. Uncinula necator) and the oomycete pathogen downy mildew (Plasmopara viticola). Currently, grapegrowers rely heavily on the use of agrochemicals to minimize the potentially devastating impact of these pathogens on grape yield and quality. The wild North American grapevine species Muscadinia rotundifolia was recognized as early as 1889 to be resistant to both powdery and downy mildew. We have now mapped resistance to these two mildew pathogens in M. rotundifolia to a single locus on chromosome 12 that contains a family of seven TIR‐NB‐LRR genes. We further demonstrate that two highly homologous (86% amino acid identity) members of this gene family confer strong resistance to these unrelated pathogens following genetic transformation into susceptible Vitis vinifera winegrape cultivars. These two genes, designated r esistance to P lasmopara v iticola (MrRPV1) are the first resistance genes to be cloned from a grapevine species. Both MrRUN1 and MrRPV1 were found to confer resistance to multiple powdery and downy mildew isolates from France, North America and Australia; however, a single powdery mildew isolate collected from the south‐eastern region of North America, to which M. rotundifolia is native, was capable of breaking MrRUN1‐mediated resistance. Comparisons of gene organization and coding sequences between M. rotundifolia and the cultivated grapevine V. vinifera at the MrRUN1/MrRPV1 locus revealed a high level of synteny, suggesting that the TIR‐NB‐LRR genes at this locus share a common ancestor.     

WAFL, a new protein involved in regulation of early endocytic transport at the intersection of actin and microtubule dynamics

We have previously identified a new gene with sequence homology to the WASP-family of actin regulators denoted WAFL (WASP and FKBP-like). Here we report a possible biological function for WAFL, by demonstrating an association to early endosomes via its central coiled-coil domain. Further we show by functional and structural studies that WAFL is associated with both microtubules and the actin filament system, the two means of transport of early endosomes. In addition, WAFL interacts with WASP-interacting protein (WIP) and actin, thus linking WAFL to actin dynamics. The use of RNAi depletion of WAFL shows that WAFL-deficient cells display delayed transport of endosomal cargo. Our findings are compatible with a model whereby WAFL is involved in the transport of early endosomes at the level of transition between microfilament-based and microtubule-based movement.     

Expression of recombinant EARLI1, a hybrid proline-rich protein of Arabidopsis, in Escherichia coli and its inhibition effect to the growth of fungal pathogens and Saccharomyces cerevisiae

EARLI1 is an Arabidopsis gene with pleiotropic effects previously shown to have auxiliary functions in protecting plants against freezing-induced cellular damage and promoting germinability under low-temperature and salinity stresses. Here we determined whether recombinant EARLI1 protein has anti-fungal activity. Recombinant EARLI1 protein lacking its signal peptide was produced in Escherichia coli BL21(DE3) using isopropyl β-d-1-thiogalactopyranoside (IPTG) induction and the prokaryotic expression vector pET28a. Expression of EARLI1 was analyzed by Western blotting and the protein was purified using affinity chromatography. Recombinant EARLI1 protein was applied to fungal cultures of Saccharomyces cerevisiae, Botrytis cinerea and Fusarium oxysporum, and membrane permeability was determined using SYTOX green. Full-length EARLI1 was expressed in S. cerevisiae from the GAL1 promoter using 2% galactose and yeast cell viability was compared to control cells. Our results indicated that application of recombinant EARLI1 protein to B. cinerea and F. oxysporum could inhibit the growth of the necrotrophic fungi. Besides, addition of the recombinant protein to liquid cultures of S. cerevisiae significantly suppressed yeast growth and cell viability by increasing membrane permeability, and in vivo expression of the secreted form of EARLI1 in S. cerevisiae also had a remarkable inhibition effect on the growth of yeast cells.