Verticillium dahliae secretory effector PevD1 induces leaf senescence by promoting ORE1-mediated ethylene biosynthesis.

Leaf senescence, the final stage of leaf development, is influenced by numerous internal and environmental signals. However, how biotic stresses such as pathogen infection regulate leaf senescence remains largely unclear. In this study, we found that the premature leaf senescence in Arabidopsis caused by the soil-borne vascular fungus Verticillium dahliae was impaired by disruption of a protein elicitor from V. dahliae 1 named PevD1. Constitutive or inducible overexpression of PevD1 accelerated Arabidopsis leaf senescence. Interestingly, a senescence-associated NAC transcription factor, ORE1, was targeted by PevD1. PevD1 could interact with and stabilize ORE1 protein by disrupting its interaction with the RING-type ubiquitin E3 ligase NLA. Mutation of ORE1 suppressed the premature senescence caused by overexpressing PevD1, whereas overexpression of ORE1 or PevD1 led to enhanced ethylene production and thereby leaf senescence. We showed that ORE1 directly binds the promoter of ACS6 and promotes its expression for mediating PevD1-induced ethylene biosynthesis. Loss-of-function of ACSs could suppress V. dahliae-induced leaf senescence in ORE1-overexpressing plants. Furthermore, we found thatPevD1 also interacts with Gossypium hirsutum ORE1 (GhORE1) and that virus-induced gene silencing of GhORE1 delays V. dahliae-triggered leaf senescence in cotton, indicating a possibly conserved mechanism in plants. Taken together, these results suggest that V. dahliae induces leaf senescence by secreting the effector PevD1 to manipulate the ORE1-ACS6 cascade, providing new insights into biotic stress-induced senescence in plants.

Wheat Pm4 resistance to powdery mildew is controlled by alternative splice variants encoding chimeric proteins.

Crop breeding for resistance to pathogens largely relies on genes encoding receptors that confer race-specific immunity. Here, we report the identification of the wheat Pm4 race-specific resistance gene to powdery mildew. Pm4 encodes a putative chimeric protein of a serine/threonine kinase and multiple C2 domains and transmembrane regions, a unique domain architecture among known resistance proteins. Pm4 undergoes constitutive alternative splicing, generating two isoforms with different protein domain topologies that are both essential for resistance function. Both isoforms interact and localize to the endoplasmatic reticulum when co-expressed. Pm4 reveals additional diversity of immune receptor architecture to be explored for breeding and suggests an endoplasmatic reticulum-based molecular mechanism of Pm4-mediated race-specific resistance.

The mechanism of sesame resistance against Macrophomina phaseolina was revealed via a comparison of transcriptomes of resistant and susceptible sesame genotypes.

BACKGROUND: Sesame (Sesamum indicum) charcoal rot, a destructive fungal disease caused by Macrophomina phaseolina (Tassi) Goid (MP), is a great threat to the yield and quality of sesame. However, there is a lack of information about the gene-for-gene relationship between sesame and MP, and the molecular mechanism behind the interaction is not yet clear. The aim of this study was to interpret the molecular mechanism of sesame resistance against MP in disease-resistant (DR) and disease-susceptible (DS) genotypes based on transcriptomics. This is the first report of the interaction between sesame and MP using this method. RESULTS: A set of core genes that response to MP were revealed by comparative transcriptomics and they were preferentially associated with GO terms such as ribosome-related processes, fruit ripening and regulation of jasmonic acid mediated signalling pathway. It is also exhibited that translational mechanism and transcriptional mechanism could co-activate in DR so that it can initiate the immunity to MP more rapidly. According to weighted gene co-expression network analysis (WGCNA) of differentially expressed gene sets between two genotypes, we found that leucine-rich repeat receptor-like kinase (LRR-RLK) proteins may assume an important job in sesame resistance against MP. Notably, compared with DS, most key genes were induced in DR such as pattern recognition receptors (PRRs) and resistance genes, indicating that DR initiated stronger pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Finally, the study showed that JA/ET and SA signalling pathways all play an important role in sesame resistance to MP. CONCLUSIONS: The defence response to MP of sesame, a complex bioprocess involving many phytohormones and disease resistance-related genes, was illustrated at the transcriptional level in our investigation. The findings shed more light on further understanding of different responses to MP in resistant and susceptible sesame.

Chitin nanofibers trigger membrane bound defense signaling and induce elicitor activity in plants.

The present study demonstrated that chitin-based nanofibers (CNFs) trigger the chitinase genes (PGIP1 and CaChi2), while elevating salicylic acid that can protect plants against pathogens. Cross-talk between this genetic induction and salicylic-acid-mediated immune response was also observed, which may arm a plant against multiple pathovars. Crab and mushroom based CNFs were synthesized by electrospinning and ball milling techniques. Plants (mung bean, Vigna radiata) (pepper, Capsicum annuum) were pre-inoculated with CNFs and treated with the pathogens Scrolotium rolfsii for pepper and Macrophomina phaseolina for mung bean and shrimp-based CNFs were used as a control. Treated plants had elevated levels of chitinase genes in response to CNFs at inoculation concentrations <10 mg/mL both in soil and media, to protect them against the pathogenic fungal disease. After 24 h of exposure to the pathogens, qRT-PCR showed genes class II chitinase gene (CaChi2) and polygalacturonase inhibitor protein 1 (PGIP1) to be up-regulated in both root and shoot at 0.1 and 1 mg/mL of inoculation, respectively. The ball milled mushroom CNFs were sufficient to trigger the membrane based enzymes with less diameter (≥15 nm) to be most efficient versus others. In vitro analysis showed IC50 of ball milled mushroom CNFs to be most efficient in limiting the growth of fungal biomass. Further trigger-like effects were prominent in reducing pathogenic fungal spread in both species.

Alleles of a wall-associated kinase gene account for three of the major northern corn leaf blight resistance loci in maize.

Northern corn leaf blight, caused by the fungal pathogen Setosphaeria turcica (anamorph Exserohilum turcicum), is one of the most devastating foliar diseases of maize (Zea mays). Four genes Ht1, Ht2, Ht3 and Htn1 represent the major sources of genetic resistance against the hemibiotrophic fungus S. turcica. Differential maize lines containing these genes also form the basis to classify S. turcica races. Here, we show that Ht2 and Ht3 are identical and allelic to the previously cloned Htn1 gene. Using a map-based cloning approach and Targeting Induced Local Lesions in Genomes (TILLING), we demonstrate that Ht2/Ht3 is an allele of the wall-associated receptor-like kinase gene ZmWAK-RLK1. The ZmWAK-RLK1 variants encoded by Htn1 and Ht2/Ht3 differ by multiple amino acid polymorphisms that particularly affect the putative extracellular domain. A diversity analysis in maize revealed the presence of dozens of ZmWAK-RLK1 alleles. Ht2, Ht3 and Htn1 have been described over decades as independent resistance loci with different race spectra and resistance responses. Our work demonstrates that these three genes are allelic, which has major implications for northern corn leaf blight resistance breeding and nomenclature of S. turcica pathotypes. We hypothesize that genetic background effects have confounded the classical description of these disease resistance genes in the past.

Determinants of defence strategies of a hibernating European bat species towards the fungal pathogen Pseudogymnoascus destructans.

Pseudogymnoascus destructans (Pd), the causative agent of white-nose syndrome in North America, has decimated bat populations within a decade. The fungus impacts bats during hibernation when physiological functions, including immune responses, are down-regulated. Studies have shown that Pd is native to Europe, where it is not associated with mass mortalities. Moreover, genomic and proteomic studies indicated that European bats may have evolved an effective immune defence, which is lacking in North American bats. However, it is still unclear which defence strategy enables European bats to cope with the pathogen. Here, we analyzed selected physiological and immunological parameters in torpid, Pd infected European greater mouse-eared bats (Myotis myotis) showing three different levels of infection (asymptomatic, mild and severe symptoms). From a subset of the studied bats we tracked skin temperatures during one month of hibernation. Contrasting North American bats, arousal patterns remained unaffected by Pd infections in M. myotis. In general, heavier M. myotis aroused more often from hibernation and showed less severe disease symptoms than lean individuals; most likely because heavy bats were capable of reducing the Pd load more effectively than lean individuals. In the blood of severely infected bats, we found higher gene expression levels of an inflammatory cytokine (IL-1ß), but lower levels of an acute phase protein (haptoglobin), reactive oxygen metabolites (ROMs) and plasma non-enzymatic antioxidant capacity (OXY) compared to conspecifics with lower levels of infection. We conclude that M. myotis, and possibly also other European bat species, tolerate Pd infections during torpor by using selected acute phase response parameters at baseline levels, yet without arousing from torpor and without synthesizing additional immune molecules.

Identification and Functional Characterization of a Novel Immunomodulatory Protein From Morchella conica SH.

A novel fungal immunomodulatory protein (FIP) was found in the precious medical and edible mushroom Morchella conica SH, defined as FIP-mco, which belongs to the FIP family. Phylogenetic analyses of FIPs from different origins were performed using Neighbor-Joining method. It was found that FIP-mco belonged to a new branch of the FIP family and may evolved from a different ancestor compared with most other FIPs. The cDNA sequence of FIP-mco was cloned and expressed in the yeast Pichia Pastoris X33. The recombinant protein of FIP-mco (rFIP-mco) was purified by agarose Ni chromatography and determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The protein rFIP-mco could significantly suppress the proliferation of A549 and HepG2 cells at the concentration of 15 and 5 µg/ml, respectively, and inhibited the migration and invasion of human A549 and HepG2 cells at the concentration of 15 and 30 µg/ml respectively in vitro. Further, rFIP-mco can significantly reduce the expression levels of TNF-α, IL-1ß, and IL-6 in the THP1 cells (human myeloid leukemia mononuclear cells). In order to explore the potential mechanism of the cytotoxicity effect of rFIP-mco on A549 and HepG2 cells, cell cycle and apoptosis assay in the two cancer cells were conducted. The results demonstrated that G0/G1 to S-phase arrest and increased apoptosis may contribute to the proliferation inhibition by rFIP-mco in the two cancer cells. Molecular mechanism of rFIP-mco's reduction effect on the inflammatory cytokines was also studied by suppression of the NF-κB signaling pathway. It showed that suppression of NF-κB signaling is responsible for the reduction of inflammatory cytokines by rFIP-mco. The results indicated the prospect of FIP-mco from M. conica SH as an effective and feasible source for cancer therapeutic studies and medical applications.

Cytokinin response induces immunity and fungal pathogen resistance, and modulates trafficking of the PRR LeEIX2 in tomato.

Plant immunity is often defined by the immunity hormones: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). These hormones are well known for differentially regulating defence responses against pathogens. In recent years, the involvement of other plant growth hormones such as auxin, gibberellic acid, abscisic acid, and cytokinins (CKs) in biotic stresses has been recognized. Previous reports have indicated that endogenous and exogenous CK treatment can result in pathogen resistance. We show here that CK induces systemic immunity in tomato (Solanum lycopersicum), modulating cellular trafficking of the pattern recognition receptor (PRR) LeEIX2, which mediates immune responses to Xyn11 family xylanases, and promoting resistance to Botrytis cinerea and Oidium neolycopersici in an SA- and ET-dependent mechanism. CK perception within the host underlies its protective effect. Our results support the notion that CK promotes pathogen resistance by inducing immunity in the host.

Early immune response against Fonsecaea pedrosoi requires Dectin-2-mediated Th17 activity, whereas Th1 response, aided by Treg cells, is crucial for fungal clearance in later stage of experimental chromoblastomycosis.

Chromoblastomycosis (CBM) is a chronic worldwide subcutaneous mycosis, caused by several dimorphic, pigmented dematiaceous fungi. It is difficult to treat patients with the disease, mainly because of its recalcitrant nature. The correct activation of host immune response is critical to avoid fungal persistence in the tissue and disease chronification. CD4+ T cells are crucial for the development of protective immunity to F. pedrosoi infection. Here, we investigated T helper cell response dynamics during experimental CBM. Following footpad injection with F. pedrosoi hyphae and conidia, T cells were skewed towards a Th17 and Th1 phenotype. The Th17 population was the main Th cell subset found in the infected area during the early stages of experimental murine CBM, followed by Th1 predominance in the later stages, coinciding with the remission phase of the disease in this experimental model. Depletion of CD25+ cells, which leads to a reduction of Treg cells in the draining lymph node, resulted in decline in fungal burden after 14 days of infection. However, fungal cells were not cleared in the later stages of the disease, prolonging CBM clinical features in those animals. IL-17A and IFN-γ neutralization hindered fungal cell elimination in the course of the disease. Similarly, in dectin-2 KO animals, Th17 contraction in the course of experimental CBM was accompanied by fungal burden decrease in the first 14 days of infection, although it did not affect disease resolution. In this study, we gained insight into T helper subsets' dynamics following footpad injections of F. pedrosoi propagules and uncovered their contribution to disease resolution. The Th17 population proved to be important in eliminating fungal cells in the early stages of infection. The Th1 population, in turn, closely assisted by Treg cells, proved to be relevant not only in the elimination of fungal cells at the beginning of infection but also essential for their complete elimination in later stages of the disease in a mouse experimental model of CBM.

Fonsecaea pedrosoi Conidia Induces Activation of Dendritic Cells and Increases CD11c+ Cells in Regional Lymph Nodes During Experimental Chromoblastomycosis.

The chromoblastomycosis is a subcutaneous mycosis with a high morbidity rate, Fonsecaea pedrosoi being the largest etiologic agent of this mycosis, usually confined to the skin and subcutaneous tissues. Rarely people get the cure, because the therapies shown to be deficient and few studies report the host-parasite relationship. Dendritic cells (DCs) are specialized in presenting antigens to naïve T lymphocytes inducing primary immune responses. Therefore, we propose to study the migratory capacity of DCs after infection with conidia of F. pedrosoi. The phenotype of DCs was evaluated using cells obtained from footpad and lymph nodes of BALB/c mice after 12, 24 and 72 h of infection. After 24 and 72 h of infection, we found a significant decrease in DCs in footpad and a significant increase in the lymph nodes after 72 h. The expression of surface markers and co-stimulatory molecules were reduced in cells obtained from footpad. To better assess the migratory capacity of DCs migration from footpad, CFSE-stained conidia were injected subcutaneously. We found that after 12 and 72 h, CD11c+ cells were increased in regional lymph nodes, leading us to believe that DCs (CD11c+) were able to phagocytic conidia present in footpad and migrated to regional lymph nodes.

Gene profiling and expression of major allergen Alt a 1 in Alternaria alternata and related members of the Pleosporaceae family.

BACKGROUND: Members of the Pleosporaceae family are known as important sources of airborne allergens which are responsible for asthma and allergic diseases. AIMS: The purpose of this study was to investigate the gene profiling and expression pattern of Alt a 1 in Alternaria alternata and other members of the Pleosporaceae family including Stemphylium botryosum, Ulocladium chartarum, Curvularia lunata, Cladosporium cladosporioides, and Epicoccum nigrum. METHODS: Alternaria alternata and related genera were cultured on Czapek-Dox broth medium at 25°C for 21 days. The presence of Alt a 1 was assessed in fungal culture filtrates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then confirmed by immunoblot analysis. Real-time PCR was carried out for quantitation of the Alt a 1 gene encoding corresponding protein at the transcriptional level using cDNA prepared from fungal RNA. RESULTS: SDS-PAGE showed protein bands ranging from 14 to 100kDa. A 14kDa band corresponding to Alt a 1 was present in A. alternata, S. botryosum and U. chartarum. The gene expression of Alt a 1 was reported in A. alternata and some other related genera. The Ct mean value recorded for A. alternata strains ranged from 24.70 to 27.84 while it was in the range 23.62-32.09 for other related taxa. No apparent transcription or expression was revealed in C. cladosporioides. CONCLUSIONS: The presence and efficient expression of Alt a 1 gene in A. alternata and other related taxa indicate that Alt a 1 protein is a major component of the secretory machinery of Pleosporaceae family members, and it may play a crucial role in its allergenicity.

Fragrant and sticky allergens from the pinewood: Cohabiting and coreacting.

BACKGROUND: Tree moss (Pseudevernia furfuracea [L.] Zopf.), a lichen growing on conifers, is a frequent fragrance sensitizer. Previous studies have shown two subgroups of tree moss-allergic patients: a group sensitized to common allergens of tree and oak moss (Evernia prunastri), and another group sensitized to colophonium-derived allergens, which may contaminate tree moss extract. OBJECTIVES: To report the results of including tree moss extract in the baseline series and discuss the clinical implications. METHODS: Tree moss extract was included in the baseline series and sensitized patients were assessed for concomitant allergy to colophonium and oak moss, and the relevance of these reactions was analyzed. RESULTS: Altogether, 22 of 632 patients (3.5%) had positive reactions to tree moss. Eight patients were sensitized to tree moss only (among fragrance allergens) and 75% had relevant reactions to colophonium. Fourteen patients were sensitized to other fragrance allergens as well and 28.5% had relevant colophonium reactions. CONCLUSIONS: The prevalence of positive tree moss reactions is high enough to justify its inclusion in the baseline series. If tree moss is not included, patients with positive colophonium reactions should be informed of possible (false) cross-reactivity to tree moss to avoid this labeled fragrance allergen.

Immunohistochemical Cross-Reactivity Between Arthrographis kalrae and Highly Pathogenic Coccidioides posadasii, Histoplasma capsulatum, and Paracoccidioides Fungal Species.

Recently, we have reported serological cross-reactivity between paracoccidioidomycosis ceti and paracoccidioidomycosis, histoplasmosis, and coccidioidomycosis. However, data on the interaction of Arthrographis kalrae with the above pathogenic fungal infections are lacking. A. kalrae is a widely occurring ascomycetous fungus; causes superficial and deep mycoses; shows thermally dependent dimorphism; and has a genomic profile related to the above-mentioned fungal species. Our study aims to investigate cross-reactivity using eight murine sera, obtained from experimental infection with two A. kalrae isolates. The murine sera were incubated with fungal cells of A. kalrae, Coccidioides posadasii, Histoplasma capsulatum, Paracoccidioides sp., and P. brasiliensis. Thirty murine sera, obtained from experimental infection with six isolates of H. capsulatum, sera from three cases of dolphin paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum sample from a healthy person with a history of coccidioidomycosis, were also incubated with A. kalrae fungal cells and the respective fungal cells that caused the infection as positive controls. Sera derived from the mice infected with A. kalrae reacted strongly when incubated with the Paracoccidioides sp., P. brasiliensis, and C. posadasii, but no positive reaction was observed against the fungal cells of H. capsulatum. The murine sera infected with three out of six isolates of H. capsulatum, and all cetacean and human serum samples reacted positively with the fungal cells of A. kalrae. The present study demonstrated serological cross-reactions among A. kalrae infection, coccidioidomycosis, paracoccidioidomycosis, paracoccidioidomycosis ceti, and histoplasmosis.

Gene profiling and expression of major allergen Alt a 1 in Alternaria alternata and related members of the Pleosporaceae family / Perfil de expresión génica del alérgeno Alt a 1 mayoritario en Alternaria alternata y otros taxones próximos de la familia Pleosporaceae

Background: Members of the Pleosporaceae family are known as important sources of airborne allergens which are responsible for asthma and allergic diseases. Aims: The purpose of this study was to investigate the gene profiling and expression pattern of Alt a 1 in Alternaria alternata and other members of the Pleosporaceae family including Stemphylium botryosum, Ulocladium chartarum, Curvularia lunata, Cladosporium cladosporioides, and Epicoccum nigrum. Methods: Alternaria alternata and related genera were cultured on Czapek-Dox broth medium at 25°C for 21 days. The presence of Alt a 1 was assessed in fungal culture filtrates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then confirmed by immunoblot analysis. Real-time PCR was carried out for quantitation of the Alt a 1 gene encoding corresponding protein at the transcriptional level using cDNA prepared from fungal RNA. Results: SDS-PAGE showed protein bands ranging from 14 to 100 kDa. A 14kDa band corresponding to Alt a 1 was present in A. alternata, S. botryosum and U. chartarum. The gene expression of Alt a 1 was reported in A. alternata and some other related genera. The Ct mean value recorded for A. alternata strains ranged from 24.70 to 27.84 while it was in the range 23.62-32.09 for other related taxa. No apparent transcription or expression was revealed in C. cladosporioides. Conclusions: The presence and efficient expression of Alt a 1 gene in A. alternata and other related taxa indicate that Alt a 1 protein is a major component of the secretory machinery of Pleosporaceae family members, and it may play a crucial role in its allergenicity

Antecedentes: Los miembros de la familia Pleosporaceae son una fuente importante de alérgenos aéreos causantes de asma y enfermedades alérgicas. Objetivos El objetivo de este trabajo fue estudiar el perfil de expresión génica de la proteína Alt a 1 en Alternaria alternata y otros miembros de la familia Pleosporaceae, entre las cuales pueden citarse Stemphylium botryosum, Ulocladium chartarum, Curvularia lunata, Cladosporium cladosporioides y Epicoccum nigrum. Métodos: Alternaria alternata y otros géneros relacionados se cultivaron en caldo Czapek-Dox a 25°C durante 21 días. La existencia de Alt a 1 en los filtrados de los cultivos se evaluó mediante electroforesis en gel de poliacrilamida con dodecilsulfato sódico (SDS-PAGE) para después confirmarla mediante inmunotransferencia. Se realizó RCP en tiempo real para la cuantificación de la transcripción del gen responsable (Alt a 1) utilizando ADNc a partir del ARN del hongo. Resultados: Mediante SDS-PAGE se visualizaron bandas de proteínas de 14 a 100 kDa. La banda de 14 kDa, correspondiente a Alt a 1, estaba presente en A. alternata, S. botryosum y U. chartarum. Se detectó expresión génica de Alt a 1 en A. alternata y otros géneros relacionados. El valor medio de Ct registrado en los aislamientos de A. alternata varió entre 24,70 y 27,84; en otros taxones cercanos, el intervalo estuvo entre 23,62 y 32,09. No se detectó transcripción o expresión en C. cladosporioides. Conclusiones: La presencia del gen Alt a 1 y su expresión en A. alternata y otros taxones próximos indica que la proteína Alt a 1 es uno de los componentes principales del mecanismo secretorio de los miembros de la familia Pleosporaceae y puede desempeñar un papel fundamental en su capacidad alergénica

Virally-vectored vaccine candidates against white-nose syndrome induce anti-fungal immune response in little brown bats (Myotis lucifugus).

White-nose syndrome (WNS) caused by the fungus, Pseudogymnoascus destructans (Pd) has killed millions of North American hibernating bats. Currently, methods to prevent the disease are limited. We conducted two trials to assess potential WNS vaccine candidates in wild-caught Myotis lucifugus. In a pilot study, we immunized bats with one of four vaccine treatments or phosphate-buffered saline (PBS) as a control and challenged them with Pd upon transfer into hibernation chambers. Bats in one vaccine-treated group, that received raccoon poxviruses (RCN) expressing Pd calnexin (CAL) and serine protease (SP), developed WNS at a lower rate (1/10) than other treatments combined (14/23), although samples sizes were small. The results of a second similar trial provided additional support for this observation. Bats vaccinated orally or by injection with RCN-CAL and RCN-SP survived Pd challenge at a significantly higher rate (P = 0.01) than controls. Using RT-PCR and flow cytometry, combined with fluorescent in situ hybridization, we determined that expression of IFN-γ transcripts and the number of CD4 + T-helper cells transcribing this gene were elevated (P < 0.10) in stimulated lymphocytes from surviving vaccinees (n = 15) compared to controls (n = 3). We conclude that vaccination with virally-vectored Pd antigens induced antifungal immunity that could potentially protect bats against WNS.

A symptomless hypovirus, CHV4, facilitates stable infection of the chestnut blight fungus by a coinfecting reovirus likely through suppression of antiviral RNA silencing.

Field-collected US strain C18 of Cryphonectria parasitica, the chestnut blight fungus, was earlier reported to be infected by a double-stranded RNA virus, mycoreovirus 2 (MyRV2). Next-generation sequencing has revealed co-infection of C18 by a positive-strand RNA virus, hypovirus 4 (CHV4). The current molecular and genetic analyses showed interesting commensal interactions between the two viruses. CHV4 facilitated the stable infection and enhanced vertical transmission of MyRV2, which was readily lost during subculturing and showed reduced vertical transmission in single infections. Deletion of a key antiviral RNA silencing gene, dcl2, in isolate C18 increased stability of MyRV2 in single infections. The ability of CHV4 to facilitate stable infection with MyRV2 appears to be associated with the inhibitory effect of CHV4 on RNA silencing via compromising the induction of transcriptional up-regulation of dcl2. These results suggest that natural infection of isolate C18 by MyRV2 in the field was facilitated by CHV4 co-infection.

Enhancing disease resistance in poplar through modification of its natural defense pathway.

KEY MESSAGE: Modification of the poplar defense pathway through pathogen-induced expression of an amphibian host defense peptide modulates plant innate immunity and confers robust and reliable resistance against a major poplar pathogen, Septoria musiva. Host defense peptides (HDPs), also known as cationic antimicrobial peptides, represent a diverse group of small membrane-active molecules that are part of the innate defense system of their hosts against pathogen invasion. Here we describe a strategy for development of poplar plants with enhanced HDP production and resistance to the commercially significant fungal pathogen Septoria musiva. The naturally occurring linear amphipathic α-helical HDP dermaseptin B1, which has 31 residues and originated from the skin secretion of arboreal frogs, was N-terminally modified (MsrA2) and evaluated in vitro for antifungal activity and phytotoxicity. The MsrA2 peptide inhibited germination of S. musiva conidia at physiologically relevant low micromolar concentrations that were non-toxic to poplar protoplasts. The nucleotide sequence of MsrA2, optimized for expression in plants, was introduced into the commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry (NM6) via Agrobacterium-mediated transformation. Transgene expression was regulated by the pathogen-inducible poplar promoter win3.12T, a part of the poplar innate defense system. Most importantly, the induced accumulation of MsrA2 peptide in poplar leaves was sufficient to confer resistance against S. musiva. The antifungal resistance of plants with high MsrA2 expression and MsrA2 accumulation was strong and reproducible, and without deleterious effects on plant growth and development. These results provide an insight into development of new technologies for engineering durable disease resistance against major pathogens of poplar and other plants.

Early-Onset Invasive Infection Due to Corynespora cassiicola Associated with Compound Heterozygous CARD9 Mutations in a Colombian Patient.

PURPOSE: CARD9 deficiency is an inborn error of immunity that predisposes otherwise healthy humans to mucocutaneous and invasive fungal infections, mostly caused by Candida, but also by dermatophytes, Aspergillus, and other fungi. Phaeohyphomycosis are an emerging group of fungal infections caused by dematiaceous fungi (phaeohyphomycetes) and are being increasingly identified in patients with CARD9 deficiency. The Corynespora genus belongs to phaeohyphomycetes and only one adult patient with CARD9 deficiency has been reported to suffer from invasive disease caused by C. cassiicola. We identified a Colombian child with an early-onset, deep, and destructive mucocutaneous infection due to C. cassiicola and we searched for mutations in CARD9. METHODS: We reviewed the medical records and immunological findings in the patient. Microbiologic tests and biopsies were performed. Whole-exome sequencing (WES) was made and Sanger sequencing was used to confirm the CARD9 mutations in the patient and her family. Finally, CARD9 protein expression was evaluated in peripheral blood mononuclear cells (PBMC) by western blotting. RESULTS: The patient was affected by a large, indurated, foul-smelling, and verrucous ulcerated lesion on the left side of the face with extensive necrosis and crusting, due to a C. cassiicola infectious disease. WES led to the identification of compound heterozygous mutations in the patient consisting of the previously reported p.Q289* nonsense (c.865C > T, exon 6) mutation, and a novel deletion (c.23_29del; p.Asp8Alafs10*) leading to a frameshift and a premature stop codon in exon 2. CARD9 protein expression was absent in peripheral blood mononuclear cells from the patient. CONCLUSION: We describe here compound heterozygous loss-of-expression mutations in CARD9 leading to severe deep and destructive mucocutaneous phaeohyphomycosis due to C. cassiicola in a Colombian child.

Immuno-impedimetric Biosensor for Onsite Monitoring of Ascospores and Forecasting of Sclerotinia Stem Rot of Canola.

Sclerotinia stem rot, caused by the fungal pathogen Sclerotinia sclerotiorum, is a destructive disease of canola and many other broadleaf crops. The primary inoculum responsible for initiating Sclerotinia epidemics is airborne ascospores released from the apothecia of sclerotia. Timely detection of the presence of airborne ascospores can serve as an early-warning system for forecasting and management of the disease. A major challenge is to develop a portable and automated device which can be deployed onsite to detect and quantify the presence of minute quantities of ascospores in the air and serves as a unit in a network of systems for forecasting of the epidemic. In this communication, we present the development of an impedimetric non-Faradaic biosensor based on anti-S. sclerotiorum polyclonal antibodies as probes to selectively capture the ascospores and sense their binding by an impedance based interdigitated electrode which was found to directly and unambiguously correlate the number of ascospores on sensor surface with the impedance response.