Showing papers in "Molecular Plant Pathology in 2006"

Sclerotinia sclerotiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen.

Abstract: Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic fungal pathogen causing disease in a wide range of plants. This review summarizes current knowledge of mechanisms employed by the fungus to parasitize its host with emphasis on biology, physiology and molecular aspects of pathogenicity. In addition, current tools for research and strategies to combat S. sclerotiorum are discussed. Taxonomy: Sclerotinia sclerotiorum (Lib.) de Bary: kingdom Fungi, phylum Ascomycota, class Discomycetes, order Helotiales, family Sclerotiniaceae, genus Sclerotinia. Identification: Hyphae are hyaline, septate, branched and multinucleate. Mycelium may appear white to tan in culture and in planta. No asexual conidia are produced. Long-term survival is mediated through the sclerotium; a pigmented, multi-hyphal structure that can remain viable over long periods of time under unfavourable conditions for growth. Sclerotia can germinate to produce mycelia or apothecia depending on environmental conditions. Apothecia produce ascospores, which are the primary means of infection in most host plants. Host range: S. sclerotiorum is capable of colonizing over 400 plant species found worldwide. The majority of these species are dicotyledonous, although a number of agriculturally significant monocotyledonous plants are also hosts. Disease symptoms: Leaves usually have water-soaked lesions that expand rapidly and move down the petiole into the stem. Infected stems of some species will first develop dark lesions whereas the initial indication in other hosts is the appearance of water-soaked stem lesions. Lesions usually develop into necrotic tissues that subsequently develop patches of fluffy white mycelium, often with sclerotia, which is the most obvious sign of plants infected with S. sclerotiorum.

Physiology and molecular aspects of Verticillium wilt diseases caused by V. dahliae and V. albo-atrum.

Abstract: Introduction: Verticillium spp. are soil-borne plant pathogens responsible for Verticillium wilt diseases in temperate and subtropical regions; collectively they affect over 200 hosts, including many economically important crops. There are currently no fungicides available to cure plants once they are infected. Taxonomy: Kingdom: Fungi, phylum: Ascomycota, subphylum, Pezizomycotina, class: Sordariomycetes, order: Phyllachorales, genus: Verticillium. Host range and disease symptoms: Over 200 mainly dicotyledonous species including herbaceous annuals, perennials and woody species are host to Verticillium diseases. As Verticillium symptoms can vary between hosts, there are no unique symptoms that belong to all plants infected by this fungus. Disease symptoms may comprise wilting, chlorosis, stunting, necrosis and vein clearing. Brown vascular discoloration may be observed in stem tissue cross-sections. Pathogenicity: Verticillium spp. have been reported to produce cell-wall-degrading enzymes and phytotoxins that all have been implicated in symptom development. Nevertheless, evidence for a crucial role of toxins in pathogenicity is inconsistent and therefore not generally accepted. Microsclerotia and melanized mycelium play an important role in the disease cycle as they are a major inoculum source and are the primary long-term survival structures. Resistance: Different defence responses in the prevascular and the vascular stage of Verticillium wilt diseases determine resistance. Although resistance physiology is well established, the molecular processes underlying this physiology remain largely unknown. Resistance against Verticillium largely depends on the isolation of the fungus in contained parts of the xylem tissues followed by subsequent elimination of the fungus. Although genetic resistance has been described in several plant species, only one resistance locus against Verticillium has been cloned to date.

Xanthomonas oryzae pathovars: model pathogens of a model crop.

Abstract: SUMMARY Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola cause bacterial blight and bacterial leaf streak of rice (Oryza sativa), which constrain production of this staple crop in much of Asia and parts of Africa. Tremendous progress has been made in characterizing the diseases and breeding for resistance. X. oryzae pv. oryzae causes bacterial blight by invading the vascular tissue, while X. oryzae pv. oryzicola causes bacterial leaf streak by colonizing the parenchyma. In rice there are 29 major genes for resistance to bacterial blight, but so far only a few quantitative resistance loci for bacterial leaf streak. Over 30 races of X. oryzae pv. oryzae have been reported. Both pathogens exhibit genetic variation among isolates. Mechanisms of pathogenesis and resistance have begun to be elucidated. Members of the AvrBs3/PthA family of transcription activator-like effectors play a major role in the virulence of X. oryzae pv. oryzae and possibly X. oryzae pv. oryzicola. Cloning of six rice resistance genes for bacterial blight and one from maize effective against bacterial leaf streak has uncovered a diversity of structure and function, some shared by genes involved in defence in animals. This article reviews research that spans a century. It also presents a perspective on challenges for sustainable control, and opportunities that interactions of X. oryzae pathovars with rice present as models for understanding fundamental aspects of bacterial pathogenesis of plants and plant disease resistance, as well as other aspects of plant and microbial biology, with implications also for animal innate immunity.

A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice.

Abstract: SUMMARY The transient assay system based on mesophyll or cultured cell-derived protoplasts has been exploited in several plant species and has become a powerful tool for rapid gene functional analysis and biochemical manipulations. However, the system has not been widely used in rice owing to the difficulties in large-scale isolation of viable rice protoplasts from leaves or suspension-cultured cells. Here, we describe a significantly improved method to isolate a large number of protoplasts from stem and sheath tissues of both young and mature plants. High-level coexpression of multiple constructs and efficient suppression of exogenous and endogenous genes were observed in the stem- and sheath-derived protoplasts. A transient green fluorescent protein and luciferase-based reporter system for defence-related genes expression analysis has been established, which is useful for screening and characterizing genes involved in rice defence signalling pathways. Furthermore, a protoplast-based bimolecular fluorescence complementation (BiFC) system for the detection of protein-protein interactions in living rice cells was developed. The YFP complementation of two split-YFP halves mediated by homodimerization of the GUS and SPIN1, a cell-death related protein, was observed in transfected protoplasts. In combination with genetic, genomic and proteomic approaches, the established versatile protoplast transient assay system will facilitate large-scale functional analysis of defence-related genes in rice.

Involvement of trichothecenes in fusarioses of wheat, barley and maize evaluated by gene disruption of the trichodiene synthase (Tri5) gene in three field isolates of different chemotype and virulence.

Abstract: SUMMARY Fusarium graminearum is the main causative agent of Fusarium head blight on small grain cereals and of ear rot on maize. The disease leads to dramatic yield losses and to an accumulation of mycotoxins. The most dominant F. graminearum mycotoxins are the trichothecenes, with deoxynivalenol and nivalenol being the most prevalent derivatives. To investigate the involvement of trichothecenes in the virulence of the pathogen, the gene coding for the initial enzyme of the trichothecene pathway was disrupted in three field isolates, differing in chemotype and in virulence. From each isolate three individual disruption mutants were tested for their virulence on wheat, barley and maize. Despite the different initial virulence of the three wild-type progenitor strains on wheat, all disruption mutants caused disease symptoms on the inoculated spikelet, but the symptoms did not spread into other spikelets. On barley, the trichothecene deficient mutants showed no significant difference compared to the wild-type strains: all were equally aggressive. On maize, mutants derived from the NIV-producing strain caused less disease than their wild-type progenitor strain, while mutants derived from DON-producing strains caused the same level of disease as their progenitor strains. These data demonstrate that trichothecenes influence the virulence of F. graminearum in a highly complex manner, which is strongly host as well as moderately chemotype specific.

Recent insights into R gene evolution

Abstract: SUMMARY Plants are under strong evolutionary pressure to maintain surveillance against pathogens. Resistance (R) gene-dependent recognition of pathogen avirulence (Avr) determinants plays a major role in plant defence. Here we highlight recent insights into the molecular mechanisms and selective forces that drive the evolution of NB-LRR (nucleotide binding-leucine-rich repeat) resistance genes. New implications for models of R gene evolution have been raised by demonstrations that R proteins can detect cognate Avr proteins indirectly by 'guarding' virulence targets, and by evidence that R protein signalling is regulated by intramolecular interactions between different R functional domains. Comparative genomic surveys of NB-LRR diversity in different species have revealed ancient NB-LRR lineages that are unequally represented among plant taxa, consistent with a Birth and Death Model of evolution. The physical distribution of NB-LRRs in plant genomes indicates that tandem and segmental duplication are important factors in R gene proliferation. The majority of R genes reside in clusters, and the frequency of recombination between clustered genes can vary strikingly, even within a single cluster. Biotic and abiotic factors have been shown to increase the frequency of recombination in reporter transgene-based assays, suggesting that external stressors can affect genome stability. Fitness penalties have been associated with some R genes, and population studies have provided evidence for maintenance of ancient R allelic diversity by balancing selection. The available data suggest that different R genes can follow strikingly distinct evolutionary trajectories, indicating that it will be difficult to formulate universally applicable models of R gene evolution.

TasHyd1, a new hydrophobin gene from the biocontrol agent Trichoderma asperellum, is involved in plant root colonization

Abstract: SUMMARY A hydrophobin-like clone (TasHyd1) was isolated during a PCR differential mRNA display analysis conducted on Trichoderma asperellum mycelia interacting with plant roots. The open reading frame encodes a 145-amino-acid protein showing similarity to Pbhyd1, a Class I hydrophobin from the dimorphic fungus Paracoccidioides brasiliensis. TasHyd1 expression was detected in planta up to 5 days after Trichoderma root inoculation. TasHyd1 is constitutively expressed at low levels in mycelia in young cultures but gene expression is not detected in sporulating hyphae or in non-germinating spores. Carbon limitation stimulates expression of TasHyd1 whereas nitrogen or phosphate starvation down-regulate expression. TasHyd1 fused to an HA tag was over-expressed in Trichoderma and the protein was detected with an anti-HA antibody in the trifluoroacetic-acid-soluble fraction of mycelial cell walls. Over-expressor mutants were not affected in their mycoparasitic activity when tested in vitro against the plant pathogen Rhizoctonia solani and retained root colonization capacity comparable with that of the wild-type. TasHyd1 deletion mutants had no significant reduction in in vitro mycoparasitic activity but were altered in their wettability and were severely impaired in root attachment and colonization. These phenotypes were recovered by complementation of TasHyd1, indicating that the protein is a new hydrophobin that contributes to Trichoderma interaction with the plant.

Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola and P. quercina in symptomatic leaves by multiplex real-time PCR

Abstract: SUMMARY New species of Phytophthora such as Phytophthora ramorum, P. kernoviae and P. quercina together with P. citricola are plant pathogens which impact on forest health, natural ecosystem stability and international trade. A real-time multiplex PCR approach based on TaqMan PCR was developed to simultaneously identify and detect these four Phytophthora species. Specific primers and probes labelled with FAM (P. ramorum), Yakima Yellow (P. kernoviae), Rox (P. citricola) and Cy5 (P. quercina) were designed in different regions of the ras-related protein (Ypt1) gene. A new set of Black Hole Quenchers (BHQ), which dissipate energy as heat rather than fluorescence, were utilized. The method proved to be highly specific in tests with target DNA from 72 Phytophthora isolates (35 species). For all pathogens, the detection limit was 100 fg of target DNA and was not improved utilizing a nested approach to provide a first round of amplification with Phytophthora spp.-specific primers. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficients ranged from 0.947 to 0.996) and were not affected by the presence of plant extracts, indicating the appropriateness of the method for qualitative and quantitative analyses. Two universal primers and a TaqMan probe were also developed to evaluate the quality and quantity of extracted DNA and to avoid false negatives. The reliability of the entire procedure was assessed using both artificially and naturally infected leaves of a range of plant species. The method, combined with a rapid procedure for DNA extraction, proved to be rapid, reliable, sensitive and cost effective as multiple pathogens were detected within the same plant extract by using different primer/probe combinations.

Stagonospora nodorum: cause of stagonospora nodorum blotch of wheat

Abstract: Stagonospora nodorum is an important pathogen of wheat and related cereals, causing both a leaf and glume blotch. This review summarizes recent advances in our understanding of taxonomy, control and pathogenicity of this species. Taxonomy: Stagonospora (syn. Septoria) nodorum (Berk.) Castell. and Germano [teleomorph: Phaeosphaeria (syn. Leptosphaeria) nodorum (Mull.) Hedjar.], kingdom Fungi, phylum Ascomycota, subphylum Euascomycota, class Dothideomycetes, order Pleosporales, family Phaeosphaeriaceae, genus Phaeosphaeria, species nodorum. Host range: Wheat, Triticum aestivum, T. durum, Triticale, are the main hosts but other cereals and wild grasses have been reported to harbour S. nodorum. Disease symptoms are lens-shaped necrotic lesions on leaves, girdling necrosis on stems (especially the nodes, hence ‘nodorum’) and lesions on glumes. Mature lesions produce pycnidia scattered throughout the lesions, especially as tissue senesces.

Signalling pathways connecting mycotoxin production and sporulation.

Abstract: SUMMARY Mycotoxin contamination of food and feed presents a serious food safety issue on a global scale, causing tremendous yield and economic losses. These toxins, produced largely by members of the genera Aspergillus and Fusarium, represent a subset of the impressive array of secondary metabolites produced by filamentous fungi. Some secondary metabolites are associated temporally and functionally with sporulation. In Aspergillus and Fusarium, sporulation and mycotoxin production are both regulated by G protein signalling pathways. G protein signalling pathways commonly regulate fungal development, stress response and expression of virulence traits. In addition, fungal development is influenced by external factors. Among these are lipids, and in particular, oxylipin signals, which may be derived from either the fungus or infected seeds. Regardless of origin, oxylipins have the potential to elicit profound changes in both sporulation and mycotoxin production in the fungus. Signal transduction via G protein signalling pathways represents one mechanism by which oxylipin signals might elicit these changes. Therefore, in this review we integrate discussion of oxylipin signals and of G protein signalling cascades as regulators of fungal development.

Proteomic and genetic approaches to identifying defence‐related proteins in rice challenged with the fungal pathogen Rhizoctonia solani

Abstract: SUMMARY Sheath blight, caused by the fungus Rhizoctonia solani , is a major disease of rice world-wide, but little is known about the host response to infection. The objective of this study was to identify proteins and DNA markers in resistant and susceptible rice associated with response to infection by R. solani . Replicated two-dimensional polyacrylamide gel electrophoresis experiments were conducted to detect proteins differentially expressed under inoculated and non-inoculated conditions. Tandem mass spectra analysis using electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS) was carried out for protein identification with the NCBI non-redundant protein database. Seven proteins were increased after inoculation in both susceptible and resistant plants. Six of the seven proteins were identified with presumed antifungal, photosynthetic and proteolytic activities. An additional 14 proteins were detected in the response of the resistant line. Eleven of the 14 proteins were identified with presumed functions relating to antifungal activity, signal transduction, energy metabolism, photosynthesis, molecular chaperone, proteolysis and antioxidation. The induction of 3- β -hydroxysteroid dehydrogenase/isomerase was detected for the first time in resistant rice plants after pathogen challenge, suggesting a defensive role of this enzyme in rice against attack by R. solani . The chromosomal locations of four induced proteins were found to be in close physical proximity to genetic markers for sheath blight resistance in two genetic mapping populations. The proteomic and genetic results from this study indicate a complex response of rice to challenge by R. solani that involves simultaneous induction of proteins from multiple defence pathways.

A novel link between tomato GRAS genes, plant disease resistance and mechanical stress response.

Abstract: SUMMARY Members of the GRAS family of transcriptional regulators have been implicated in the control of plant growth and development, and in the interaction of plants with symbiotic bacteria. Here we examine the complexity of the GRAS gene family in tomato (Solanum lycopersicum) and investigate its role in disease resistance and mechanical stress. A large number of tomato ESTs corresponding to GRAS transcripts were retrieved from the public database and assembled in 17 contigs of putative genes. Expression analysis of these genes by real-time RT-PCR revealed that six SlGRAS transcripts accumulate during the onset of disease resistance to Pseudomonas syringae pv. tomato. Further analysis of two selected family members showed that their transcripts preferentially accumulate in tomato plants in response to different avirulent bacteria or to the fungal elicitor EIX, and their expression kinetics correlate with the appearance of the hypersensitive response. In addition, transcript levels of eight SlGRAS genes, including all the Pseudomonas-inducible family members, increased in response to mechanical stress much earlier than upon pathogen attack. Accumulation of SlGRAS transcripts following mechanical stress was in part dependent on the signalling molecule jasmonic acid. Remarkably, suppression of SlGRAS6 gene expression by virus-induced gene silencing impaired tomato resistance to P. syringae pv. tomato. These results support a function for GRAS transcriptional regulators in the plant response to biotic and abiotic stress.

mlo‐based powdery mildew immunity: silver bullet or simply non‐host resistance?

Abstract: SUMMARY Durability and effectiveness against all genetic variants of a microbial species are hallmarks of so-called plant 'non-host' resistance. Highly effective immunity of monocotyledonous barley against the fungal powdery mildew pathogen, which is conferred by loss-of-function mutant alleles of the barley Mlo locus, likewise is a durable and broad-spectrum type of resistance. Although this was long considered as being a barley-specific phenomenon, recent findings indicate that mlo resistance can also occur in the distantly related dicotyledonous species Arabidopsis thaliana. Shared histological and phytopathological characteristics plus a conserved requirement for a set of genes in Arabidopsis mlo and non-host powdery mildew resistance indicate a potential common mechanism for these two seemingly distinct types of immunity.

Selection for increased cyproconazole tolerance in Mycosphaerella graminicola through local adaptation and in response to host resistance.

Abstract: SUMMARY Sterol demethylation inhibitors (DMIs) represent one of the largest groups of systemic fungicides that have been used to control agriculturally important fungal pathogens. Knowledge regarding the evolution of fungicide resistance in agricultural ecosystems is fragmentary and a better understanding of the processes driving the development of DMI resistance in populations of fungal pathogens is needed by plant pathologists and the agrochemical industry. We considered some of these processes using approaches based on molecular population and quantitative genetics. Five Mycosphaerella graminicola populations sampled from unsprayed wheat fields on four continents were assayed for eight restriction fragment length polymorphism (RFLP) markers and their level of tolerance to cyproconazole. DMI fungicides such as cyproconazole inhibit the enzyme eburicol 14-alpha-demethylase. The gene encoding this target, CYP51, was sequenced for all isolates. We found unimodal, continuous variations in cyproconazole tolerance among the M. graminicola isolates sampled from individual fields, consistent with a polygenic mode of inheritance. We also found that population differentiation for cyproconazole tolerance (Q(ST)) among the five M. graminicola populations was significantly higher than the corresponding population differentiation for neutral RFLP markers (G(ST)), suggesting that selection for cyproconazole tolerance in the Swiss population has already led to local adaptation that can be seen even in an unsprayed population. The Swiss population displayed the highest level of tolerance to cyproconazole, in addition to a lower than expected quantitative variation in fungicide tolerance and a skewed distribution, indicating that selection had increased the overall tolerance of this population. Further analysis with DNA sequencing showed that the population from Switzerland was dominated by isolates with several point mutations and a 6-bp deletion in CYP51. This deletion and one of the point mutations were previously related to increased resistance in field isolates. The fungal population from Oregon sampled from an unsprayed resistant host cultivar displayed the same gene diversity in RFLP loci but higher cyproconazole tolerance and quantitative variation in tolerance than the fungal population from the same field sampled from an unsprayed susceptible host cultivar.

Population genetic structure of Plasmopara viticola after 125 years of colonization in European vineyards.

Abstract: SUMMARY To examine the within- and among-population genetic structure of Plamopara viticola oosporic populations in Europe, 8991 lesions from 32 vineyard plots were collected and analysed. Four multi-allelic microsatellite markers were used to genotype the pathogen. All populations had high levels of gene and genotypic diversity. Most populations were in Hardy–Weinberg equilibrium and thus randomly mating. Among P. viticola populations, significant low to moderate genetic differentiation was observed, even between geographically close populations. This genetic differentiation was also evident in the neighbour-joining phylogenetic genetic distance tree, showing clear substructure and distinguishing mainly five clusters based on geographical origin. Significant isolation by distance was found in central European P. viticola populations, suggesting a step-wise migration model. No significant isolation by distance was found within Greek populations, most probably owing to natural geographical barriers such as the sea and mountains, as well as the frequent population bottlenecks occurring in these populations, preventing natural migration among populations. The high variability of P. viticola provides explanation for its successful infestation of the heterogeneous European vineyards in the last 125 years after its introduction.

Extensin over-expression in Arabidopsis limits pathogen invasiveness.

Abstract: SUMMARY The function of the cell wall protein extensin has been the subject of much speculation since it was first isolated over 40 years ago. In order to investigate the role of extensins in plant defence, we used the gain-of-function strategy to generate transgenic Arabidopsis plants over-expressing the EXT1 extensin gene. These were infected with the virulent bacterial pathogen Pseudomonas syringae DC3000 and symptom development was monitored. Lesions on the transgenics were on average five-fold smaller than those on the wild-type, did not increase in area over the time period of infection, accumulated a small bacterial load and showed very little chlorosis outside the lesion boundary. By contrast, lesions on the wild-type were large, spread to over 50% of the leaf area, continued to increase in size over the time course of the infection, accumulated a bacterial load 100-fold higher than that found in the transgenics, and showed a large chlorotic area outside the lesion boundary. SEM of lesions showed no evidence of bacteria at the lesion boundary in the extensin-over-expressing transgenics, whereas bacteria were always seen at the lesion boundary on the wild-type. Analysis of transgenics carrying an EXT1-GUS promoter-reporter fusion showed expression of GUS in a ring around the boundary of the lesion. Basal defences and signal transduction pathways involved in plant defence were not perturbed in the transgenics, as shown by the analysis of the expression of PR1 and PDF1.2 genes. These results show that extensin over-expression limits pathogen invasiveness.

The global nitrogen regulator, FNR1, regulates fungal nutrition-genes and fitness during Fusarium oxysporum pathogenesis

Abstract: SUMMARY Fusarium oxysporum is a soil-borne pathogen that infects plants through the roots and uses the vascular system for host ingress. Specialized for this route of infection, F. oxysporum is able to adapt to the scarce nutrient environment in the xylem vessels. Here we report the cloning of the F. oxysporum global nitrogen regulator, Fnr1, and show that it is one of the determinants for fungal fitness during in planta growth. The Fnr1 gene has a single conserved GATA-type zinc finger domain and is 96% and 48% identical to AREA-GF from Gibberella fujikuroi, and NIT2 from Neurospora crassa, respectively. Fnr1 cDNA, expressed under a constitutive promoter, was able to complement functionally an N. crassa nit-2(RIP) mutant, restoring the ability of the mutant to utilize nitrate. Fnr1 disruption mutants showed high tolerance to chlorate and reduced ability to utilize several secondary nitrogen sources such as amino acids, hypoxanthine and uric acid, whereas growth on favourable nitrogen sources was not affected. Fnr1 disruption also abolished in vitro expression of nutrition genes, normally induced during the early phase of infection. In an infection assay on tomato seedlings, infection rate of disruption mutants was significantly delayed in comparison with the parental strain. Our results indicate that FNR1 mediates adaptation to nitrogen-poor conditions in planta through the regulation of secondary nitrogen acquisition, and as such acts as a determinant for fungal fitness during infection.

The MAP kinase-encoding gene MgFus3 of the non-appressorium phytopathogen Mycosphaerella graminicola is required for penetration and in vitro pycnidia formation

Abstract: In eukaryotes, a family of serine/threonine protein kinases known as mitogen-activated protein kinases (MAPKs) is involved in the transduction of a variety of extracellular signals and in the regulation of growth and development. We identified a MAPK-encoding gene in Mycosphaerella graminicola strain IPO323 with high homology to the orthologous Fus3 gene of Saccharomyces cerevisiae and designated it MgFus3. Early colony development of the MgFus3 mutants during in vitro growth was similar to those of the wild-type and ectopic controls, but at the later stages of growth MgFus3 mutants did not become melanized, showed altered polarized growth and were unable to produce aerial mycelia. The MgFus3 mutants were non-pathogenic, and detailed microscopic analyses revealed that they failed to colonize the mesophyll tissue owing to the inability to penetrate stomata. Unlike the wild-type strain, MgFus3 mutants were unable to differentiate pycnidia on plant-derived media. Thus, in addition to the crucial role of MgFus3 in the regulation of penetration, it may also be involved in regulating asexual fructification. Hence, MgFus3 can be regarded as a multifunctional pathogenicity factor of M. graminicola

Agroinfection-based high-throughput screening reveals specific recognition of INF elicitins in Solanum

Abstract: SUMMARY We adapted and optimized the use of the Agrobacterium tumefaciens binary PVX expression system (PVX agroinfection) to screen Solanum plants for response to pathogen elicitors and applied the assay to identify a total of 11 clones of Solanum huancabambense and Solanum microdontum, out of 31 species tested, that respond to the elicitins INF1, INF2A and INF2B of Phytophthora infestans. Prior to this study, response to INF elicitins was only known in Nicotiana spp. within the Solanaceae. The identified S. huancabambense and S. microdontum clones also exhibited hypersensitivity-like cell death following infiltration with purified recombinant INF1, INF2A and INF2B, thereby validating the screening protocol. Comparison of INF elicitin activity revealed that Nicotiana plants responded to significantly lower concentrations than Solanum, suggesting variable levels of sensitivity to INF elicitins. We exploited natural variation in response to INF elicitins in the identified Solanum accessions to evaluate the relationship between INF recognition and late blight resistance. Interestingly, several INF-responsive Solanum plants were susceptible to P. infestans. Also, an S. microdontum xSolanum tuberosum (potato) population that segregates for INF response was generated but failed to identify a measurable contribution of INF response to resistance. These results suggest that in Solanum, INF elicitins are recognized as general elicitors and do not have a measurable contribution to disease resistance.

Re-organization of the cytoskeleton and endoplasmic reticulum in the Arabidopsis pen1-1 mutant inoculated with the non-adapted powdery mildew pathogen, Blumeria graminis f. sp. hordei.

Abstract: SUMMARY Plant cells attacked by microorganisms rapidly translocate cytoplasm to the site of pathogen penetration, a response that usually involves rearrangement of actin microfilaments. In this study, we monitored re-organization of green fluorescent protein (GFP)-labelled actin microfilaments, microtubules and endoplasmic reticulum (ER) during infection by the powdery mildew pathogen Blumeria graminis f. sp. hordei in non-host Arabidopsis and in the Arabidopsis penetration 1-1 (pen1-1) mutant, which shows increased penetration susceptibility to non-adapted pathogens. Comparison of pen1-1 with wild-type Arabidopsis showed that the actin, microtubule and ER networks all responded in the pen1-1 mutant as they do in wild-type plants. Actin microfilaments became focused on the penetration site and ER accumulated at the penetration site while the overall arrangement of microtubule arrays was largely unaffected. These results indicate that the block in vesicle secretion conferred by the pen1-1 mutation does not interfere with cytoplasmic aggregation or recruitment of actin or ER to the infection site. In the pen1-1 mutant, the higher rate of successful penetration by the non-adapted pathogen results in an increased incidence of hypersensitive cell death. In dying cells, the structure of the ER was rapidly destroyed, in contrast to actin microfilaments and microtubules which remained for a longer time after the initiation of cell death. In Arabidopsis with GFP-tagged tubulin, fluorescent vesicle-like structures appeared near the cell surface during the initiation of cell death. In both wild-type and pen1-1 mutant plants, in cells surrounding the dying cell, bundles of actin microfilaments focused on the anticlinal walls adjacent to the dead cell and ER accumulated in the cortical cytoplasm near the dead cell. These observations suggest that the neighbouring, non-infected cells use actin-based transport to secrete material at the surface adjacent to the dead cell. They also indicate that disruption of secretion (in the pen1-1 mutant) does not inhibit transmission of signals from the dying cells to their neighbours.

Genetic and proteomic analysis of the role of luxS in the enteric phytopathogen, Erwinia carotovora.

Abstract: SUMMARY Erwinia carotovora is a Gram-negative phytopathogen that is an important cause of soft rot disease, including stem and tuber rot in potatoes. Quorum sensing is the process by which bacteria detect their population density and regulate gene expression accordingly. Quorum sensing, an important example of intercellular communication, involves the production and detection of chemical signal molecules. The enzyme LuxS is responsible for the production of Autoinducer-2 (AI-2), a molecule that has been implicated in quorum sensing in many bacterial species. In this study, the role of luxS in Erwinia carotovora ssp. carotovora strain ATTn10 and Erwinia carotovora ssp. atroseptica SCRI1043 has been examined. Both strains have been shown to produce luxS-dependent extracellular AI-2 activity and the phenotypes of defined luxS mutants in these strains have been characterized. Inactivation of luxS in Er. carotovora was found to have a strain-dependent impact on the intracellular proteome (using two-dimensional difference in gel electrophoresis), secreted proteins, motility and virulence in planta. No link was detected with the N-acyl-l-homoserine lactone quorum sensing system in these organisms. Although the molecular mechanism(s) of luxS regulation in Erwinia remain to be determined, this is the first report of any luxS-dependent phenotypes in a plant pathogen.

Characterization of Arabidopsis thaliana–Fusarium graminearum interactions and identification of variation in resistance among ecotypes

Abstract: SUMMARY Fusarium graminearum causes fusarium head blight (FHB) of wheat and other cereals. Resistance of wheat to FHB is incomplete and the defence mechanisms involved are poorly understood owing to the complex genome and long life cycle of the host. A robust and reproducible bioassay system was established for the study of interactions between F. graminearum and Arabidopsis by wound inoculation of detached leaves embedded in agar medium. Amendment of the inoculum with deoxynivalenol enhanced symptom development and conidial production. Colonization of leaf tissue was by both inter- and intracellular growth and extensive cell death was observed ahead of advancing hyphae indicating the presence of phytotoxic compounds or host-initiated programmed cell death. Expression of defence response genes associated with jasmonic acid/ethylene and salicylic acid pathways indicated that both pathways were activated in response to F. graminearum with expression of the jasmonic acid/ethylene pathways being more enhanced than that of the salicylic acid pathway, as expected for a necrotrophic pathogen. A large variation in resistance among Arabidopsis ecotypes was revealed. The ecotype Col-0 was significantly more resistant than Ler using both the detached leaf and a detached flower assay. Foliar resistance was shown to be controlled largely by a single genetic factor on chromosome 4 near the RFLP marker O6455. The detached leaf assay described herein provides a means to enable fundamental studies on the defence mechanisms of Arabidopsis in response to F. graminearum.

Nuclear import and export of plant virus proteins and genomes

Abstract: Nuclear import and export are crucial processes for any eukaryotic cell, as they govern substrate exchange between the nucleus and the cytoplasm. Proteins involved in the nuclear transport network are generally conserved among eukaryotes, from yeast and fungi to animals and plants. Various pathogens, including some plant viruses, need to enter the host nucleus to gain access to its replication machinery or to integrate their DNA into the host genome; the newly replicated viral genomes then need to exit the nucleus to spread between host cells. To gain the ability to enter and exit the nucleus, these pathogens encode proteins that recognize cellular nuclear transport receptors and utilize the host's nuclear import and export pathways. Here, we review and discuss our current knowledge about the molecular mechanisms by which plant viruses find their way into and out of the host cell nucleus.

Nitrogen controls in planta expression of Cladosporium fulvum Avr9 but no other effector genes

Abstract: During growth on its host tomato, the apoplast-colonizing fungal pathogen Cladosporium fulvum secretes several effector proteins. The expression of the Avr9 gene encoding one of these effector proteins has previously been shown to be strongly induced in vitro during nitrogen deprivation. This led to the hypothesis that expression of additional effector genes in C. fulvum could be triggered by nitrogen starvation conditions that are encountered in the host. We now show that expression of most effectors is not affected by varying levels of nitrogen supplementation in vitro. In addition, we demonstrate that the nitrogen response regulator Nrf1 only regulates Avr9 expression during infection of the host, whereas none of the other known effectors is significantly controlled by this transcription factor in planta. Deletion of Nrf1, but not of Avr9, significantly reduces C. fulvum virulence. Therefore, it is concluded that Nrf1 controls, in addition to Avr9, unidentified effector genes that are required for full virulence of C. fulvum

Novel elicitin‐like proteins isolated from the cell wall of the biocontrol agent Pythium oligandrum induce defence‐related genes in sugar beet

Abstract: SUMMARY We previously reported that cell wall protein fractions (CWPs) of the biocontrol agent Pythium oligandrum have elicitor properties in sugar beet and wheat. Here we have examined the effect of treatment with the D-type of CWP, a fraction that contains two major forms (POD-1 and POD-2), on the induction of defence-related genes in sugar beet. Using PCR-based cDNA library subtraction, we identified five genes that were highly expressed in response to CWP treatment. The five genes are probably of oxalate oxidase-like germin (OxOLG), glutathione S-transferase (GST), 5-enol-pyruvylshikimate-phosphate synthase (EPSPS), phenylalanine ammonia-lyase (PAL) and aspartate aminotransferase (AAT). In addition, we purified and characterized POD-1 and POD-2 and found that POD-1 induced all five genes, whereas POD-2 induced three of the genes, but not OxOLG or GST. A sugar beet bioassay indicated that CWP, POD-1 and POD-2 are each sufficient to induce resistance to sugar beet seedling disease caused by Aphanomyces cochlioides. Although carbohydrate analyses indicated that POD proteins were glycoproteins with similar carbohydrate compositions, containing approximately 15.0% carbohydrate by weight, their peptide portions have elicitor activity. Furthermore, cDNAs of POD-1 and POD-2 proteins were cloned, and the deduced amino acid sequences were found to be 82.9% identical. Characterization of their molecular structures indicated that they have an elicitin domain followed by a C-terminal domain with a high frequency of Ser, Thr, Ala and Pro, which is structurally similar to class III elicitins. However, phylogenetic analysis with 22 representative elicitin and elicitin-like proteins showed that POD-1 and POD-2 are distinct from previously defined elicitin and elicitin-like proteins. Therefore, POD-1 and POD-2 are novel oomycete cell wall elicitin-like glycoproteins.

Comparative genomic analysis of phytopathogenic fungi using expressed sequence tag (EST) collections.

Abstract: SUMMARY We describe the analysis of 57 727 unique expressed sequence tags (ESTs) from 15 species of phytopathogenic and three species of saprophytic fungi. This resource is held within the COGEME phytopathogen EST database (http://cogeme.ex.ac.uk/). Comparative analysis was performed to investigate the differences between pathogenic and free-living fungi based on a substantial collection of expressed gene sequences and available, completed fungal genome sequences. We report that the expressed gene inventories of pathogenic fungi were not significantly more similar to each other than to those of free-living filamentous fungi. As expected, however, filamentous fungi as a group share more sequences in common than with the free-living yeast species Saccharomyces cerevisiae. Interestingly, ESTs of the obligate biotrophic fungus Blumeria graminis f. sp. hordei were more dissimilar to those of all other fungal species assessed, having a lower number of sequences in common with filamentous ascomycetes studied to date and also possessing a larger proportion of unisequences of unknown function. Our analysis of ESTs in the COGEME database enabled identification of a set of functional groups of genes that are more highly represented in the genomes of pathogenic fungi than non-pathogenic species.

Expression of barley BAX Inhibitor-1 in carrots confers resistance to Botrytis cinerea.

Abstract: SUMMARY BAX Inhibitor-1 (BI-1) is a protein that controls heterologous BAX-induced cell death, the hypersensitive reaction and abiotic stress-induced cell death in plants. When over-expressed in epidermal cells of barley, barley BI-1 induces susceptibility to the biotrophic fungal pathogen Blumeria graminis. When we expressed barley BI-1 in carrot susceptible to the necrotrophic fungus Botrytis cinerea, we obtained BI-1-mediated resistance to fungus-induced leaf cell death and less fungal spreading on the leaves. Barley BI-1 also mediated resistance to Chalara elegans in carrot roots. The results support the idea that cell death inhibition is an applicable approach to control cell-death-inducing pathogens in crop plants.

Laser capture microdissection: a novel approach to microanalysis of plant–microbe interactions

Abstract: Gene expression studies are often carried out at the whole organism, organ or tissue levels. The different cell types present in most tissue exhibit different patterns of gene expression. This limits analyses because results obtained represent an average of the activities of the different cell types, and may lead to masking of genes of interest that are specifically expressed in a particular cell type. The recent development of laser capture microdissection (LCM) now enables target cells to be isolated from complex tissues and allows analysis of specific cell types that represent the in vivo state at the time of sample extraction. LCM has been applied to analyse plant tissues in a number of studies. This review illustrates the application of LCM in studies on gene expression profiling and proteomics, and also in research on plant–microbe interactions.

Peach latent mosaic viroid: not so latent.

Abstract: SUMMARY Taxonomy: Peach latent mosaic viroid (PLMVd) is the type species of the genus Pelamoviroid within the family Avsunviroidae of chloroplastic viroids with hammerhead ribozymes. Physical properties: A small circular RNA of 336–351 nt (differences in size result from the absence or presence of certain insertions) adopting a branched conformation stabilized by a pseudoknot between two kissing loops. This particular conformation is most likely responsible for the insolubility of PLMVd in highly saline conditions (in which other viroids adopting a rod-like conformation are soluble). Both polarity strands are able to form hammerhead structures and to self-cleave during replication as predicted by these ribozymes. Biological properties: Although most infections occur without conspicuous symptoms, certain PLMVd isolates induce leaf mosaics, blotches and in the most extreme cases albinism (peach calico, PC), flower streaking, delays in foliation, flowering and ripening, deformations and decolorations of fruits, which usually present cracked sutures and enlarged roundish stones, bud necrosis, stem pitting and premature ageing of the trees, which also adopt a characteristic growing pattern (open habit). The molecular determinant for PC has been mapped at a 12–14-nt insertion that folds into a hairpin capped by a U-rich loop present only in certain variants. PLMVd is horizontally transmitted by the propagation of infected buds and to a lesser extent by pruning tools and aphids, but not by pollen; the viroid is not vertically transmitted through seed. Interesting features: This provides a suitable system for studying how a minimal non-protein-coding catalytic RNA replicates (subverting a DNA-dependent RNA polymerase to transcribe an RNA template), moves, interferes with the metabolism of its host (inciting specific symptoms and a defensive RNA silencing response) and evolves following a quasi-species model characterized by a complex spectrum of variants.

Transient over‐expression of barley BAX Inhibitor‐1 weakens oxidative defence and MLA12‐mediated resistance to Blumeria graminis f.sp. hordei

Abstract: SUMMARY BAX Inhibitor-1 (BI-1) is a conserved cell death suppressor protein. In barley, BI-1 (HvBI-1) expression is induced upon powdery mildew infection and when over-expressed in epidermal cells of barley, HvBI-1 induces susceptibility to the biotrophic fungal pathogen Blumeria graminis. We co-expressed mammalian pro-apoptotic BAX together with HvBI-1, and the mammalian BAX antagonist BCL-X(L) in barley epidermal cells. BAX expression led to cessation of cytoplasmic streaming and collapse of the cytoplasm while co-expression of HvBI-1 and BCL-X(L) partially or completely, respectively, rescued cells from BAX lethality. When B. graminis was attacking epidermal cells, a green fluorescent protein fusion of HvBI-1 accumulated at the site of attempted penetration and was also present around haustoria. Over-expression of HvBI-1 in epidermal cells weakened a cell-wall-associated local hydrogen peroxide burst in a resistant mlo-mutant genotype and supported haustoria accommodation in race-specifically resistant MLA12-barley. HvBI-1 is a cell death regulator protein of barley with the potential to suppress host defence reactions.