Cellulose Binding Elicitor Lectin Research Articles

First report of the NA2 clonal lineage of the sudden oak death pathogen, Phytophthora ramorum, infecting tanoak in Oregon forests.

Phytophthora ramorum Werres, de Cock & Man in't Veld, causal agent of sudden oak death (SOD) and ramorum leaf blight, is comprised of four clonal lineages in its invasive ranges of North America and Europe (Grünwald et al. 2012, Van Poucke et al. 2012). Of these, three - the NA1, NA2, and EU1 lineages - are found in U.S. nurseries, but only two, the NA1 and EU1 lineages, have been found infecting trees in North American forests (Grünwald et al. 2012, 2016). In the spring of 2021, tanoak (Notholithocarpus densiflorus Manos, Cannon & Oh) displaying symptoms consistent with SOD were detected north of Port Orford (Curry County, Oregon). Symptoms were canopy dieback and blackened petiole and stem lesions on tanoak sprouts. The pathogen isolated on PAR (CMA plus 200 ml/L ampicillin, 10 mg/L rifamycin, 66.7 mg/L PCNB) selective media was determined to be P. ramorum based on characteristic morphology of hyphae, sporangia, and chlamydospores (Werres et al. 2001). Positive identification as P. ramorum was obtained with a lineage-specific LAMP assay targeting an NA2 orphan gene, indicating the presence of the NA2 lineage. NA2 was confirmed by sequencing a portion of the cellulose binding elicitor lectin (CBEL) gene using CBEL5U and CBEL6L primers (Gagnon et al. 2014). Sequences (GenBank accessions MZ733981 and MZ733982) were aligned against reference sequences for all lineages (Gagnon et al. 2014) confirming the presence of NA2. Lineage determination as NA2 was further confirmed at eleven SSR loci (ILVOPrMS145, PrMS39, PrMS9C3, ILVOPrMS79, KI18, KI64, PrMS45, PrMS6, ILVOPrMS131, KI82ab, and PrMS43) using the methods of Kamvar et al. (2015). We completed Koch's postulates using potted tanoaks, wound-inoculated at the midpoint of 1-year old stems with either hyphal plugs or non-colonized agar (n=4 per treatment). Tanoaks were maintained in a growth chamber (20°C-day / 18°C-night temperatures) with regular watering and an 18-photoperiod using F32T8 fluorescent bulbs (Phillips, Eindhoven, The Netherlands). After 7 days, brown to black lesions 1.2 to 2.9 cm in length were observed on the inoculated stems, from which P. ramorum was subsequently re-isolated; no symptoms were observed on the controls, and no pathogens were recovered when plating the wound sites in PAR. This is the first detection of the NA2 lineage causing disease in forests worldwide. The outbreak was found on private and public lands in forests typical to the SOD outbreak in Oregon (mixed conifer and tanoak), and was 33 km north of the closest known P. ramorum infestation. Follow-up ground surveys on adjacent lands have identified over 100 P. ramorum-positive tanoak trees, from which additional NA2 isolates have been recovered from bole cankers. NA2 is thought to be more aggressive than the NA1 lineage (Elliott et al. 2011), which has been present in Curry County since the mid-1990s (Goheen et al. 2017). Eradication of the NA2 lineage is being pursued to slow its further spread and prevent overlap with existing NA1 and EU1 populations. The repeated introductions of novel lineages into the western United States native plant communities highlights the vulnerability of this region to Phytophthora establishment, justifying continued monitoring for P. ramorum in nurseries and forests. References • Elliott, M, et al. 2011. For. Path. 41:7. https://doi.org/10.1111/j.1439-0329.2009.00627.x • Gagnon, M.-C., et al. 2014. Can. J. Plant Pathol. 36:367. https://doi.org/10.1080/07060661.2014.924999 • Goheen, E.M., et al. 2017. For. Phytophthoras 7:45. https://doi: 10.5399/osu/fp.7.1.4030 • Grünwald, N. J., et al. 2012. Trends Microbiol. 20:131. https://doi.org/10.1016/j.tim.2011.12.006 • Grünwald, N. J., et al. 2016. Plant Dis. 100:1024. https://doi.org/10.1094/PDIS-10-15-1169-PDN • Kamvar, Z.N. et al. 2015. Phytopath. 105:982. https://doi.org/10.1094/PHYTO-12-14-0350-FI • Van Poucke, K., et al. 2012. Fungal Biol. 116:1178. https://doi.org/10.1016/j.funbio.2012.09.003 • Werres, S., et al. 2001. Mycol. Res. 105: 1155. https://doi.org/10.1016/S0953-7562(08)61986-3.

Trichoderma Counteracts the Challenge of Phytophthora nicotianae Infections on Tomato by Modulating Plant Defense Mechanisms and the Expression of Crinkler, Necrosis-Inducing Phytophthora Protein 1, and Cellulose-Binding Elicitor Lectin Pathogenic Effectors.

Decoding the mechanisms of plant defense against plant pathogens in a scenario where antagonistic activity and the plant growth-promoting effects of useful organisms intervene simultaneously is a new frontier of plant pathology. Here, we demonstrated that (i) two selected strains of Trichoderma asperellum and Trichoderma atroviride promoted tomato (Solanum lycopersicum) growth and reduced the severity of disease caused by the oomycete Phytophthora nicotianae and (ii) the genetic patterns of the components of the experimental model system tomato–Trichoderma spp.–P. nicotianae were differentially expressed. The beneficial effects in both the promotion of the growth of host plant and the biological control of the pathogen by two selected strains of different Trichoderma species were tested both in planta and in vitro. In both respects, T. atroviride demonstrated to be more effective than T. asperellum. Additionally, the simultaneous transcriptional reprogramming of several plant defense-related genes, pathogen effectors, and mycoparasitism-related genes in tomato, P. nicotianae, and Trichoderma spp., respectively, was evaluated during the three-component interaction. Results support the hypothesis that Trichoderma spp. elicit the expression of plant defense-related genes. As expected, a mycoparasitism-related gene was significantly up-regulated in Trichoderma-colonizing tomato plants infected by P. nicotianae. Finally, a marked up-regulation of the genes encoding two necrosis-inducing effectors was observed in P. nicotianae infecting tomato plants colonized by Trichoderma. In conclusion, this study is a contribution toward understanding the genetic pathways related with the ability of Trichoderma spp. to counteract the challenge of P. nicotianae infections on tomato. Additionally, the experiments revealed the beneficial effects in the tomato growth promotion of a new T. atroviride strain and its good antagonistic effectiveness in the biological control of root and crown rot incited by P. nicotianae, confirming that Trichoderma spp. can be a powerful tool in integrated pest management strategies of Phytophthora diseases of horticultural crops.

First Report of the NA2 Clonal Lineage of Phytophthora ramorum in Indiana

The oomycete pathogen Phytophthora ramorum is the causal agent of ramorum leaf blight and sudden oak death (Rizzo et al. 2002). P. ramorum is known to cause leaf blight and stem dieback on nursery hosts including Rhododendron. The disease typically results in dark, brownish lesions on leaves or stems, which can cause wilting and death of the plant (Grunwald et al. 2008; Werres et al. 2001). Since the emergence of the disease, a federal quarantine was established to prevent the spread of the pathogen, and nurseries that export P. ramorum hosts are subject to a federally mandated certification program for interstate export (Grunwald et al. 2012). This disease has had a significant impact on the U.S. nursery industry via quarantine regulations imposed on nurseries infested with the pathogen. The pathogen has been introduced at least three times into the Western United States as clonal lineages NA1, NA2, and EU1 (Grunwald et al. 2009, 2019; Ivors et al. 2006). In the spring and summer of 2019, USDA-APHIS reported that a shipment of potentially P. ramorum-infested plants was delivered to several Eastern and Midwestern states. Rhododendron leaves from numerous counties in Indiana, showing characteristic necrotic leaf blight symptoms, were sampled by Indiana Department of Natural Resources (IDNR) nursery inspectors between April 18 and June 3, 2019. These samples were initially screened for the presence of Phytophthora at the Purdue Plant and Pest Diagnostic Laboratory using an ELISA test (Agdia). Subsamples from tissue producing a positive ELISA result were forwarded to the Michigan State University Plant & Pest Diagnostics laboratory for P. ramorum-specific PCR testing, which was later confirmed by USDA CPHST. Leaf surfaces of P. ramorum-positive samples were surface disinfested to remove contaminating organisms by washing vigorously in 50 ml of 70% ethanol for 10 s followed by three rinses in sterile water. Washed leaves were blotted dry on sterile paper towels. From each leaf, 5-mm leaf discs were punched out of the leading edge of leaf lesions, and the resulting leaf discs were submerged in selective V8 medium amended with pimaricin, ampicillin, and rifampicin in Petri dishes. Petri dishes were incubated at 20°C until growth was present. A plug of growth from the leading edge of the resulting colony was transferred to fresh V8 medium containing a 47-mm 0.4-micron polycarbonate filter (Nucleopore) and incubated for approximately 8 days at 20°C. DNA was extracted by the Center for Genome Research and Biocomputing at Oregon State University using the Omega BioTek Plant DNA DS kit (M1130), and part of the cellulose binding elicitor lectin gene (CBEL) was amplified and sequenced with primers CBEL5U and CBEL6L (Gagnon et al. 2014). Sequences were aligned with the CBEL reference sequences of EU1 (KF679685), EU2 (KF679716), NA1 (EU688908), and NA2 (KF679712) (Gagnon et al. 2014). All 26 of the Indiana isolates were unambiguously classified as belonging to the NA2 clonal lineage (GenBank accessions MN601787 to MN601812). This is the first report of the NA2 clonal lineage outside of British Columbia, Washington, and California, indicating that this pathogen was most likely moved West to East. Prior documented lineages detected obtained from P. ramorum-infected plants in Eastern receiving states have only harbored the NA1 clonal lineage (Goss et al. 2009, 2011). As a result of these detections, the IDNR ordered destruction of more than 6,100 Rhododendron plants at retail outlets in Indiana.

First Report of Phytophthora ramorum Lineage EU1 Infecting Douglas Fir and Grand Fir in Oregon

HomePlant DiseaseVol. 102, No. 2First Report of Phytophthora ramorum Lineage EU1 Infecting Douglas Fir and Grand Fir in Oregon PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Phytophthora ramorum Lineage EU1 Infecting Douglas Fir and Grand Fir in OregonJ. M. LeBoldus, K. L. Sondreli, W. Sutton, P. Reeser, S. Navarro, A. Kanaskie, and N. J. GrünwaldJ. M. LeBoldus†Corresponding author: J. M. LeBoldus; E-mail: E-mail Address: jared.leboldus@science.oregonstate.eduSearch for more papers by this author, K. L. SondreliSearch for more papers by this author, W. SuttonSearch for more papers by this author, P. ReeserSearch for more papers by this author, S. NavarroSearch for more papers by this author, A. KanaskieSearch for more papers by this author, and N. J. Grünwaldhttp://orcid.org/0000-0003-1656-7602Search for more papers by this authorAffiliationsAuthors and Affiliations J. M. LeBoldus † , Oregon State University, Department of Botany and Plant Pathology and Forest Engineering, Resources and Management, Corvallis, OR 97331 K. L. Sondreli W. Sutton P. Reeser , Oregon State University, Department of Botany and Plant Pathology, Corvallis, OR 97331 S. Navarro A. Kanaskie , Oregon Department of Forestry, Private Forests Program, Forest Health Section, Salem, OR 97310 N. J. Grünwald , Horticultural Crops Research Laboratory, USDA Agricultural Research Service, Corvallis, OR 97330. Published Online:21 Nov 2017https://doi.org/10.1094/PDIS-05-17-0681-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Sudden oak death (SOD) is caused by the introduced oomycete pathogen Phytophthora ramorum S. Werres, A. W. A. M. de Cock & W. A. Man in’t Veld. This fungus-like organism has four clonal lineages: NA1, NA2, EU1, and EU2 (Grünwald et al. 2016; Prospero et al. 2007). Until recently, the NA1 lineage was the only clonal lineage of P. ramorum reported in wildland forests in the western United States. In contrast, EU1, NA1, and NA2 have all been found in U.S. nurseries (Grünwald et al. 2012). In the winter of 2015, a symptomatic Notholithocarpus densiflorus Manos, Cannon & S.H.Oh (tanoak) was identified during a SOD helicopter survey in Curry County, OR. P. ramorum was isolated from symptomatic bark tissue. Subsequently, the isolate was determined to be of the EU1 lineage based on 14 microsatellite loci (Grünwald et al. 2016). Continued monitoring of the area in 2016 and 2017 has identified symptomatic Abies grandis (Douglas ex D. Don) Lindl. (grand fir; n = 3) and Pseudotsuga menziesii (Mirb.) Franco (Douglas fir; n = 2) saplings growing near infected tanoak trees in the mixed conifer forest of Curry County, OR. Symptoms with shoot blight for these species were similar to those described in the literature, including wilting and dieback of new shoots, brown discoloration of needles, and needle loss on young shoots. Isolations were made by plating surface-sterilized tip dieback tissue from A. grandis and P. menziesii on a Phytophthora-selective medium. Based on the presence of chlamydospores, characteristic hyphae, and sporangial morphology, the isolates were identified as P. ramorum. DNA was extracted from hyphae, and a portion of the cellulose binding elicitor lectin (CBEL) gene was amplified and sequenced using the CBEL5U and CBEL6L primers (Gagnon et al. 2014). The sequences of the unknown lineage were aligned to sequences of CBEL for NA1, NA2, EU1, and EU2 using the Staden package in GAP version 4.11.2. The lineage of the isolates from A. grandis and P. menziesii (GenBank accession nos. MF918374 and MF918375, respectively) had 100% identity to the EU1 reference sequences (GenBank nos. EU688952 and EF117945). To satisfy Koch’s postulates, three branches of A. grandis and P. menziessii were inoculated with the original EU1 isolates of P. ramorum from those hosts. Ten days after inoculation, the same pathogen was reisolated from symptomatic stem tissue of both tree species. The EU1 lineage is considered more aggressive than the NA1 lineage and is of opposite mating type to NA1, thus potentially resulting in establishment of sexual populations.

Extracellular Recognition of Oomycetes during Biotrophic Infection of Plants.

Extracellular recognition of pathogens by plants constitutes an important early detection system in plant immunity. Microbe-derived molecules, also named patterns, can be recognized by pattern recognition receptors (PRRs) on the host cell membrane that trigger plant immune responses. Most knowledge on extracellular pathogen detection by plants comes from research on bacterial and fungal pathogens. For oomycetes, that comprise some of the most destructive plant pathogens, mechanisms of extracellular pattern recognition have only emerged recently. These include newly recognized patterns, e.g., cellulose-binding elicitor lectin, necrosis and ethylene-inducing peptide 1-like proteins (NLPs), and glycoside hydrolase 12, as well as their receptors, e.g., the putative elicitin PRR elicitin response and the NLP PRR receptor-like protein 23. Immunity can also be triggered by the release of endogenous host-derived patterns, as a result of oomycete enzymes or damage. In this review we will describe the types of patterns, both pathogen-derived exogenous and plant-derived endogenous ones, and what is known about their extracellular detection during (hemi-)biotrophic oomycete infection of plants.

Transcriptome analysis of the entomopathogenic oomycete Lagenidium giganteum reveals putative virulence factors.

A combination of 454 pyrosequencing and Sanger sequencing was used to sample and characterize the transcriptome of the entomopathogenic oomycete Lagenidium giganteum. More than 50,000 high-throughput reads were annotated through homology searches. Several selected reads served as seeds for the amplification and sequencing of full-length transcripts. Phylogenetic analyses inferred from full-length cellulose synthase alignments revealed that L giganteum is nested within the peronosporalean galaxy and as such appears to have evolved from a phytopathogenic ancestor. In agreement with the phylogeny reconstructions, full-length L. giganteum oomycete effector orthologs, corresponding to the cellulose-binding elicitor lectin (CBEL), crinkler (CRN), and elicitin proteins, were characterized by domain organizations similar to those of pathogenicity factors of plant-pathogenic oomycetes. Importantly, the L. giganteum effectors provide a basis for detailing the roles of canonical CRN, CBEL, and elicitin proteins in the infectious process of an oomycete known principally as an animal pathogen. Finally, phylogenetic analyses and genome mining identified members of glycoside hydrolase family 5 subfamily 27 (GH5_27) as putative virulence factors active on the host insect cuticle, based in part on the fact that GH5_27 genes are shared by entomopathogenic oomycetes and fungi but are underrepresented in nonentomopathogenic genomes. The genomic resources gathered from the L. giganteum transcriptome analysis strongly suggest that filamentous entomopathogens (oomycetes and fungi) exhibit convergent evolution: they have evolved independently from plant-associated microbes, have retained genes indicative of plant associations, and may share similar cores of virulence factors, such as GH5_27 enzymes, that are absent from the genomes of their plant-pathogenic relatives.

Real-time PCR assay to distinguish Phytophthora ramorum lineages using the cellulose binding elicitor lectin (CBEL) locus

Phytophthora ramorum is a pathogenic oomycete that causes sudden oak death in the Western USA and sudden larch death in the UK. Until recently, three genetically divergent clonal lineages of P. ramorum were known (EU1, NA1 and NA2), named according to the continent on which they were first detected. In 2009, a fourth lineage named EU2 was discovered in the UK. Sequencing and microsatellite genotyping revealed that the EU2 lineage is genetically distinct from all other lineages. Allele-specific oligonucleotide-PCR (ASO-PCR) assays using real-time PCR were developed in this study, allowing for the identification of the EU2 lineage. Also, a combination of ASO-PCR assays targeting the cellulose binding elicitor lectin (CBEL) locus was validated to rapidly identify all four lineages. Sequencing of the CBEL locus revealed eight single nucleotide polymorphisms (SNPs) that distinguished EU2 from the other three lineages. Two ASO-PCR assays were developed from these SNPs, providing the ability to rapidly identify EU2 individuals relative to EU1, NA1 and NA2 individuals. These new assays were combined with two existing assays targeting the same locus to allow rapid and simple identification of all four lineages. Blind tests performed on a panel of representative samples revealed diagnostic profiles unique to each lineage. These markers can be used with diseased field samples, making them well suited for routine procedures in diagnostic laboratories to identify P. ramorum.

Pathogen-associated molecular pattern-triggered immunity and resistance to the root pathogen Phytophthora parasitica in Arabidopsis.

The cellulose binding elicitor lectin (CBEL) of the genus Phytophthora induces necrosis and immune responses in several plant species, including Arabidopsis thaliana. However, the role of CBEL-induced responses in the outcome of the interaction is still unclear. This study shows that some of CBEL-induced defence responses, but not necrosis, required the receptor-like kinase BAK1, a general regulator of basal immunity in Arabidopsis, and the production of a reactive oxygen burst mediated by respiratory burst oxidases homologues (RBOH). Screening of a core collection of 48 Arabidopsis ecotypes using CBEL uncovered a large variability in CBEL-induced necrotic responses. Analysis of non-responsive CBEL lines Ws-4, Oy-0, and Bla-1 revealed that Ws-4 and Oy-0 were also impaired in the production of the oxidative burst and expression of defence genes, whereas Bla-1 was partially affected in these responses. Infection tests using two Phytophthora parasitica strains, Pp310 and Ppn0, virulent and avirulent, respectively, on the Col-0 line showed that BAK1 and RBOH mutants were susceptible to Ppn0, suggesting that some immune responses controlled by these genes, but not CBEL-induced cell death, are required for Phytophthora parasitica resistance. However, Ws-4, Oy-0, and Bla-1 lines were not affected in Ppn0 resistance, showing that natural variability in CBEL responsiveness is not correlated to Phytophthora susceptibility. Overall, the results uncover a BAK1- and RBOH-dependent CBEL-triggered immunity essential for Phytophthora resistance and suggest that natural quantitative variation of basal immunity triggered by conserved general elicitors such as CBEL does not correlate to Phytophthora susceptibility.

The unique architecture and function of cellulose-interacting proteins in oomycetes revealed by genomic and structural analyses.

BackgroundOomycetes are fungal-like microorganisms evolutionary distinct from true fungi, belonging to the Stramenopile lineage and comprising major plant pathogens. Both oomycetes and fungi express proteins able to interact with cellulose, a major component of plant and oomycete cell walls, through the presence of carbohydrate-binding module belonging to the family 1 (CBM1). Fungal CBM1-containing proteins were implicated in cellulose degradation whereas in oomycetes, the Cellulose Binding Elicitor Lectin (CBEL), a well-characterized CBM1-protein from Phytophthora parasitica, was implicated in cell wall integrity, adhesion to cellulosic substrates and induction of plant immunity.ResultsTo extend our knowledge on CBM1-containing proteins in oomycetes, we have conducted a comprehensive analysis on 60 fungi and 7 oomycetes genomes leading to the identification of 518 CBM1-containing proteins. In plant-interacting microorganisms, the larger number of CBM1-protein coding genes is expressed by necrotroph and hemibiotrophic pathogens, whereas a strong reduction of these genes is observed in symbionts and biotrophs. In fungi, more than 70% of CBM1-containing proteins correspond to enzymatic proteins in which CBM1 is associated with a catalytic unit involved in cellulose degradation. In oomycetes more than 90% of proteins are similar to CBEL in which CBM1 is associated with a non-catalytic PAN/Apple domain, known to interact with specific carbohydrates or proteins. Distinct Stramenopile genomes like diatoms and brown algae are devoid of CBM1 coding genes. A CBM1-PAN/Apple association 3D structural modeling was built allowing the identification of amino acid residues interacting with cellulose and suggesting the putative interaction of the PAN/Apple domain with another type of glucan. By Surface Plasmon Resonance experiments, we showed that CBEL binds to glycoproteins through galactose or N-acetyl-galactosamine motifs.ConclusionsThis study provides insight into the evolution and biological roles of CBM1-containing proteins from oomycetes. We show that while CBM1s from fungi and oomycetes are similar, they team up with different protein domains, either in proteins implicated in the degradation of plant cell wall components in the case of fungi or in proteins involved in adhesion to polysaccharidic substrates in the case of oomycetes. This work highlighted the unique role and evolution of CBM1 proteins in oomycete among the Stramenopile lineage.

Expression and purification of a biologically active Phytophthora parasitica cellulose binding elicitor lectin in Pichia pastoris

The Phytophthora parasitica cellulose-binding elicitor lectin, (CBEL), is a cell wall-localized protein playing a key role in cell wall organization and adhesion of the mycelium to cellulosic substrates. CBEL is a potent elicitor of plant immune responses and this activity is linked to its ability to bind plant cell wall components. In order to scale up the production of active CBEL, we reported here the cloning and expression of a His-tagged version of CBEL in the yeast Pichia pastoris. Selection of a high-producing P. pastoris clone and optimization of the purification procedure allowed a yield of about 2mg of pure protein per liter of culture filtrate. The identity of the recombinant protein was confirmed by western-blot analysis, N-terminal protein sequencing, and by peptide mass fingerprinting. The cellulose-binding affinity and the lectin activity of the recombinant protein were identical to the native CBEL. Its elicitor activity, tested on Arabidopsis thaliana leaves, was similar to the native CBEL protein as it displays a similar biological activity on plant immune responses inducing defense gene expression and localized necroses of the infiltrated leaf tissues. The present work suggests that P. pastoris can be a suitable host for the production of compounds active on plants or for the development of new agricultural products able to stimulate plant immunity.

CMPG1‐dependent cell death follows perception of diverse pathogen elicitors at the host plasma membrane and is suppressed by Phytophthora infestans RXLR effector AVR3a

• Little is known about how effectors from filamentous eukaryotic plant pathogens manipulate host defences. Recently, Phytophthora infestans RXLR effector AVR3a has been shown to target and stabilize host E3 ligase CMPG1, which is required for programmed cell death (PCD) triggered by INF1. We investigated the involvement of CMPG1 in PCD elicited by perception of diverse pathogen proteins, and assessed whether AVR3a could suppress each. • The role of CMPG1 in PCD events was investigated using virus-induced gene silencing, and the ability of AVR3a to suppress each was determined by transient expression of natural forms (AVR3a(KI) and AVR3a(EM)) and a mutated form, AVR3a(KI/Y147del) , which is unable to interact with or stabilize CMPG1. • PCD triggered at the host plasma membrane by Cf-9/Avr9, Cf-4/Avr4, Pto/AvrPto or the oomycete pathogen-associated molecular pattern (PAMP), cellulose-binding elicitor lectin (CBEL), required CMPG1 and was suppressed by AVR3a, but not by the AVR3a(KI/Y147del) mutant. Conversely, PCD triggered by nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins R3a, R2 and Rx was independent of CMPG1 and unaffected by AVR3a. • CMPG1-dependent PCD follows perception of diverse pathogen elicitors externally or in association with the inner surface of the host plasma membrane. We argue that AVR3a targets CMPG1 to block initial signal transduction/regulatory processes following pathogen perception at the plasma membrane.

Cellulose-binding domains: cellulose associated-defensive sensing partners?

The cellulose-binding domains (CBDs) in the Phytophthora cellulose-binding elicitor lectin (CBEL) are potent elicitors of plant defence responses. Induction of defence has also been reported in various cellulose-deficient mutants of Arabidopsis thaliana. Based on these observations, we propose a model linking cellulose alteration to defence induction. This integrates the fast increase in cytosolic calcium recorded in response to CBEL, mechano-stimulated calcium uptake mechanisms, and proteins that interact functionally with the cellulose synthase complex. In this context, CBDs emerge as new tools to decipher the signalling cascades that result from cell wall-cellulose perturbations.

Differentiation of European and North American genotypes of Phytophthora ramorum by real-time polymerase chain reaction primer extension

Phytophthora ramorum, the organism responsible for sudden oak death, causes severe damage to oak (Quercus spp.) and tanoak (Lithocarpus densiflorus) trees in California and Oregon. There are now >100 host plants that can be affected by this pathogen in nurseries and in the wild in Europe and North America. Molecular diagnosis and genotyping are important tools to monitor and prevent spread of this pathogen, to enforce quarantines, and to support eradication programs. By amplifying and sequencing DNA in a worldwide panel of strains of P. ramorum, we discovered single-nucleotide polymorphisms (SNP) in two genes (β -tubulin and cellulose binding elicitor lectin (CBEL)) that differ in the European and the North American P. ramorum isolates. Using these SNPs in diagnostic assays and distinguishing among strains of different geographic origins, we developed primer extension assays in real-time polymerase chain reaction using allele-specific oligonucleotides. All European isolates genotyped were homozygous for the SNP at position 279 in the β -tubulin gene (β- tub279 ; G/G) but heterozygous at position 858 (β- tub858 ; A/G). By contrast, all the California isolates were heterozygous for β -tub279 (T/G) but homozygous for β -tub858 (A/A). For CBEL, all European isolates were heterozygous for the SNPs at position 245 (CBEL245 ; G/C) and 412 (CBEL412 ; G/A), but the California samples were homozygous at both SNP loci CBEL245 (G/G) and CBEL412 (G/G). This assay was also used directly on DNA extracted from the ELISA lysates from field samples collected in British Columbia in 2005 by the Canadian Food Inspection Agency. Although most samples had the North American DNA profiles, there were several samples with the European profiles from the nurseries. This finding could provide information on the biology, epidemiology, etiology, host range, and populations of P. ramorum from North America and Europe and provide useful tools in P. ramorum surveys. Key words: primer extension, β- tubulin, CBEL, genotyping, allele specific oligonucleotide, SybrGreen, mating type, PCR-ASO. Le Phytophthora ramorum, l'organisme responsable de l'encre des chênes rouges ou aussi appelé la mort subite du chêne, cause de sérieux dommages aux chênes (Quercus spp.) et lithocarpes (Lithocarpus densiflorus) de la Californie et de l'Oregon. Il y a maintenant plus de 100 espèces de plantes hôtes pouvant être infectées par cet agent pathogène en Europe et en Amérique du nord, autant en pépinière qu'en nature. Le diagnostic moléculaire et le génotypage sont des outils permettant de répertorier et de prévenir la propagation de cet agent pathogène, de renforcer les mesures de quarantaines et de supporter les programmes d'éradication de la maladie. En amplifiant et en séquençant l'ADN d'une collection mondiale du P. ramorum, nous avons découvert des polymorphismes dans deux gènes (β -tubuline et CBEL « cellulose binding elicitor lectin ») pouvant différencier les isolats européens des nord- américains du P. ramorum. En utilisant ces SNP dans des tests diagnostics et en différenciant les souches de diverses origines géographiques, nous avons utilisé la méthode d'extension des amorces avec la réaction de polymerisation en chaîne (PCR) en temps-réel et des oligonucleotides spécifiques d'allèle. Tous les isolats européens génotypés étaient homozygotes pour la β- tubuline pour le SNP β -tub279 (G/G), mais hétérozygotes pour β -tub858 (A/G). Par ailleurs, tous les isolats californiens étaient hétérozygotes pour β -tub279 (T/G), mais homozygotes pour β -tub858 (A/A). Pour ce qui est de CBEL, tous les isolats européens étaient hétérozygotes pour CBEL245 (G/C) et CBEL412 (G/A), mais les échantillons de Californie étaient homozygotes aux deux SNP loci, CBEL245 (G/G) et CBEL412 (G/G). Ce test a aussi été utilisé directement sur des échantillons environnementaux d'ADN extraits de lysats d'ELISA provenant de pépinières de Colombie-Britannique dans une enquête de l'Agence canadienne d'inspection des aliments réaliseé en 2005. Presque tous ces échantillons présentaient un profile nord américain contre quelques-uns présentant un profil européen. Cette découverte est importante car elle fournit de l'information sur la biologie, l'épidémiologie, l'étiologie, la diversité d'hôtes et les populations du P. ramorum d'Europe et d'Amérique du nord. Mots-clés : extension des amorces, β- tubuline, CBEL, génotypage, oligonucléotide spécifique d'allèle, SybrGreen, type sexuel, PCR-ASO.

Cellulose Binding Domains of a Phytophthora Cell Wall Protein Are Novel Pathogen-Associated Molecular Patterns

The cellulose binding elicitor lectin (CBEL) from Phytophthora parasitica nicotianae contains two cellulose binding domains (CBDs) belonging to the Carbohydrate Binding Module1 family, which is found almost exclusively in fungi. The mechanism by which CBEL is perceived by the host plant remains unknown. The role of CBDs in eliciting activity was investigated using modified versions of the protein produced in Escherichia coli or synthesized in planta through the potato virus X expression system. Recombinant CBEL produced by E. coli elicited necrotic lesions and defense gene expression when injected into tobacco (Nicotiana tabacum) leaves. CBEL production in planta induced necrosis. Site-directed mutagenesis on aromatic amino acid residues located within the CBDs as well as leaf infiltration assays using mutated and truncated recombinant proteins confirmed the importance of intact CBDs to induce defense responses. Tobacco and Arabidopsis thaliana leaf infiltration assays using synthetic peptides showed that the CBDs of CBEL are essential and sufficient to stimulate defense responses. Moreover, CBEL elicits a transient variation of cytosolic calcium levels in tobacco cells but not in protoplasts. These results define CBDs as a novel class of molecular patterns in oomycetes that are targeted by the innate immune system of plants and might act through interaction with the cell wall.