Genetic characterization of Pepino mosaic virus isolates from Belgian greenhouse tomatoes reveals genetic recombination

Over a period of a few years, Pepino mosaic virus (PepMV) has become one of the most important viral diseases in tomato production worldwide. Infection by PepMV can cause a broad range of symptoms on tomato plants, often leading to significant financial losses. At present, five PepMV genotypes (EU, LP, CH2, US1 and US2) have been described, three of which (EU, LP and US2) have been reported in Europe. Thus far, no correlation has been found between different PepMV genotypes and the symptoms expressed in infected plants. In this paper, the genetic diversity of the PepMV population in Belgian greenhouses is studied and related to symptom development in tomato crops. A novel assay based on restriction fragment length polymorphism (RFLP) was developed to discriminate between the different PepMV genotypes. Both RFLP and sequence analysis revealed the occurrence of two genotypes, the EU genotype and the CH2 genotype, within tomato production in Belgium. Whereas no differences were observed in symptom expression between plants infected by one of the two genotypes, co-infection with both genotypes resulted in more severe PepMV symptoms. Furthermore, our study revealed that PepMV recombinants frequently occur in mixed infections under natural conditions. This may possibly result in the generation of viral variants with increased aggressivenes

OcculterCut: A comprehensive survey of AT-rich regions in fungal genomes.

We present a novel method to measure the local GC-content bias in genomes and a survey of published fungal species. The method, enacted as "OcculterCut" (https://sourceforge.net/projects/occultercut), identified species containing distinct AT-rich regions. In most fungal taxa, AT-rich regions are a signature of repeat-induced point mutation (RIP), which targets repetitive DNA and decreases GC-content though the conversion of cytosine to thymine bases. RIP has in turn been identified as a driver of fungal genome evolution, as RIP mutations can also occur in single-copy genes neighbouring repeat-rich regions. Over time RIP perpetuates 'two speeds' of gene evolution in the GC-equilibrated and AT-rich regions of fungal genomes. In this study, genomes showing evidence of this process are found to be common, particularly among the Pezizomycotina. Further analysis highlighted differences in amino acid composition and putative functions of genes from these regions, supporting the hypothesis that these regions play an important role in fungal evolution. OcculterCut can also be used to identify genes undergoing RIP-assisted diversifying selection, such as small, secreted effector proteins that mediate host-microbe disease interactions

Colletotrichum higginsianum extracellular LysM proteins play dual roles in appressorial function and suppression of chitin-triggered plant immunity

<p>The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large repertoire of candidate-secreted effectors containing LysM domains, but the role of such proteins in the pathogenicity of any Colletotrichum species is unknown. Here, we characterized the function of two effectors, ChELP1 and ChELP2, which are transcriptionally activated during the initial intracellular biotrophic phase of infection. Using immunocytochemistry, we found that ChELP2 is concentrated on the surface of bulbous biotrophic hyphae at the interface with living host cells but is absent from filamentous necrotrophic hyphae. We show that recombinant ChELP1 and ChELP2 bind chitin and chitin oligomers in vitro with high affinity and specificity and that both proteins suppress the chitin-triggered activation of two immune-related plant mitogen-activated protein kinases in the host Arabidopsis. Using RNAi-mediated gene silencing, we found that ChELP1 and ChELP2 are essential for fungal virulence and appressorium-mediated penetration of both Arabidopsis epidermal cells and cellophane membranes in vitro. The findings suggest a dual role for these LysM proteins as effectors for suppressing chitin-triggered immunity and as proteins required for appressorium function.</p

Effector-triggered defence against apoplastic fungal pathogens

Copyright 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license CC BY 3.0 (http://creativecommons.org/licenses/by/3.0/). hR gene-mediated host resistance against apoplastic fungal pathogens is not adequately explained by the terms pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) or effector-triggered immunity (ETI). Therefore, it is proposed that this type of resistance is termed ‘effector-triggered defence’ (ETD). Unlike PTI and ETI, ETD is mediated by R genes encoding cell surface-localised receptor-like proteins (RLPs) that engage the receptor-like kinase SOBIR1. In contrast to this extracellular recognition, ETI is initiated by intracellular detection of pathogen effectors. ETI is usually associated with fast, hypersensitive host cell death, whereas ETD often triggers host cell death only after an elapsed period of endophytic pathogen growth. In this opinion, we focus on ETD responses against foliar fungal pathogens of cropsPeer reviewe

Selected reactive oxygen species and antioxidant enzymes in common bean after Pseudomonas syringae pv. phaseolicola and Botrytis cinerea infection

Phaseolus vulgaris cv. Korona plants were inoculated with the bacteria Pseudomonas syringae pv. phaseolicola (Psp), necrotrophic fungus Botrytis cinerea (Bc) or with both pathogens sequentially. The aim of the experiment was to determine how plants cope with multiple infection with pathogens having different attack strategy. Possible suppression of the non-specific infection with the necrotrophic fungus Bc by earlier Psp inoculation was examined. Concentration of reactive oxygen species (ROS), such as superoxide anion (O2 -) and H2O2 and activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were determined 6, 12, 24 and 48 h after inoculation. The measurements were done for ROS cytosolic fraction and enzymatic cytosolic or apoplastic fraction. Infection with Psp caused significant increase in ROS levels since the beginning of experiment. Activity of the apoplastic enzymes also increased remarkably at the beginning of experiment in contrast to the cytosolic ones. Cytosolic SOD and guaiacol peroxidase (GPOD) activities achieved the maximum values 48 h after treatment. Additional forms of the examined enzymes after specific Psp infection were identified; however, they were not present after single Bc inoculation. Subsequent Bc infection resulted only in changes of H2O2 and SOD that occurred to be especially important during plant–pathogen interaction. Cultivar Korona of common bean is considered to be resistant to Psp and mobilises its system upon infection with these bacteria. We put forward a hypothesis that the extent of defence reaction was so great that subsequent infection did not trigger significant additional response

Structure-Activity Determinants in Antifungal Plant Defensins MsDef1 and MtDef4 with Different Modes of Action against Fusarium graminearum

Plant defensins are small cysteine-rich antimicrobial proteins. Their three-dimensional structures are similar in that they consist of an α-helix and three anti-parallel β-strands stabilized by four disulfide bonds. Plant defensins MsDef1 and MtDef4 are potent inhibitors of the growth of several filamentous fungi including Fusarium graminearum. However, they differ markedly in their antifungal properties as well as modes of antifungal action. MsDef1 induces prolific hyperbranching of fungal hyphae, whereas MtDef4 does not. Both defensins contain a highly conserved γ-core motif (GXCX3–9C), a hallmark signature present in the disulfide-stabilized antimicrobial peptides, composed of β2 and β3 strands and the interposed loop. The γ-core motifs of these two defensins differ significantly in their primary amino acid sequences and in their net charge. In this study, we have found that the major determinants of the antifungal activity and morphogenicity of these defensins reside in their γ-core motifs. The MsDef1-γ4 variant in which the γ-core motif of MsDef1 was replaced by that of MtDef4 was almost as potent as MtDef4 and also failed to induce hyperbranching of fungal hyphae. Importantly, the γ-core motif of MtDef4 alone was capable of inhibiting fungal growth, but that of MsDef1 was not. The analysis of synthetic γ-core variants of MtDef4 indicated that the cationic and hydrophobic amino acids were important for antifungal activity. Both MsDef1 and MtDef4 induced plasma membrane permeabilization; however, kinetic studies revealed that MtDef4 was more efficient in permeabilizing fungal plasma membrane than MsDef1. Furthermore, the in vitro antifungal activity of MsDef1, MsDef1-γ4, MtDef4 and peptides derived from the γ-core motif of each defensin was not solely dependent on their ability to permeabilize the fungal plasma membrane. The data reported here indicate that the γ-core motif defines the unique antifungal properties of each defensin and may facilitate de novo design of more potent antifungal peptides

Plutajuće matriks tablete: Dizajniranje i optimizacija kombiniranjem polimera

The purpose of the present study was to develop an optimized gastric floating drug delivery system (GFDDS) containing domperidone as a model drug. Box-Behnken design was employed in formulating the GFDDS with three polymers: hydroxypropyl methylcellulose K4M (HPMC K4M) (X1), Carbopol 934P (X2) and sodium alginate (X3), as independent variables. Floating lag time (FLT), total floating time (TFT), time required to release 50% of the drug (t50) and diffusion exponent (n) were selected as dependent variables. Seventeen formulations were prepared, dissolution data obtained was fitted to the power law and floating profiles were analyzed. HPMC loading was found to be significant for floating properties. Carbopol loading had a negative effect on floating properties but was found helpful in controlling the release rate of the drug. No significant effect of sodium alginate on floating properties was observed but it was important for gel formation. The quadratic mathematical model developed could be used to predict formulations with desired release and floating properties.Cilj rada bio je razvoj i optimizacija plutajućih sustava za isporuku lijekova u želucu (GFDDS) s domperidonom kao modelom lijeka. Box-Behnkenovo dizajniranje korišteno je u formuliranju GFDDS. Nezavisne varijable u dizajniranju bila su tri polimera: hidroksipropil metilceluloza K4M (HPMC K4M) (X1), Carbopol 934P (X2) i natrijev alginat (X3), a zavisne varijable usporeno vrijeme plutanja (FLT), ukupno vrijeme plutanja (TFT), vrijeme potrebno za oslobađanje 50% lijeka (t50) i difuzijski eksponent (n). Pripravljeno je ukupno sedamnaest formulacija. Analizirani su podaci o oslobađanju ljekovite tvari. Količina HPMC značajno utječe na svojstva plutanja, dok količina karbopola ima negativni učinak na svojstvo plutanja, ali kontrolira oslobađanje ljekovite tvari. Natrijev alginat nema značajni učinak na svojstva plutanja, ali utječe na stvaranje gela. Kvadratni matematički model može se upotrijebiti za predviđanje formulacija sa željenim profilom oslobađanja i svojstvima plutanja

Petunia Floral Defensins with Unique Prodomains as Novel Candidates for Development of Fusarium Wilt Resistance in Transgenic Banana Plants

Antimicrobial peptides are a potent group of defense active molecules that have been utilized in developing resistance against a multitude of plant pathogens. Floral defensins constitute a group of cysteine-rich peptides showing potent growth inhibition of pathogenic filamentous fungi especially Fusarium oxysporum in vitro. Full length genes coding for two Petunia floral defensins, PhDef1 and PhDef2 having unique C- terminal 31 and 27 amino acid long predicted prodomains, were overexpressed in transgenic banana plants using embryogenic cells as explants for Agrobacterium–mediated genetic transformation. High level constitutive expression of these defensins in elite banana cv. Rasthali led to significant resistance against infection of Fusarium oxysporum f. sp. cubense as shown by in vitro and ex vivo bioassay studies. Transgenic banana lines expressing either of the two defensins were clearly less chlorotic and had significantly less infestation and discoloration in the vital corm region of the plant as compared to untransformed controls. Transgenic banana plants expressing high level of full-length PhDef1 and PhDef2 were phenotypically normal and no stunting was observed. In conclusion, our results suggest that high-level constitutive expression of floral defensins having distinctive prodomains is an efficient strategy for development of fungal resistance in economically important fruit crops like banana

The interactive effects of arbuscular mycorrhiza and plant growth-promoting rhizobacteria synergistically enhance host plant defences against pathogens

Belowground interactions between plant roots, mycorrhizal fungi and plant growth-promoting rhizobacteria (PGPR) can improve plant health via enhanced nutrient acquisition and priming of the plant immune system. Two wheat cultivars differing in their ability to form mycorrhiza were (co)inoculated with the mycorrhizal fungus Rhizophagus irregularis and the rhizobacterial strain Pseudomonas putida KT2440. The cultivar with high mycorrhizal compatibility supported higher levels of rhizobacterial colonization than the low compatibility cultivar. Those levels were augmented by mycorrhizal infection. Conversely, rhizobacterial colonization of the low compatibility cultivar was reduced by mycorrhizal arbuscule formation. Single inoculations with R. irregularis or P. putida had differential growth effects on both cultivars. Furthermore, while both cultivars developed systemic priming of chitosan-induced callose after single inoculations with R. irregularis or P. putida, only the cultivar with high mycorrhizal compatibility showed a synergistic increase in callose responsiveness following co-inoculation with both microbes. Our results show that multilateral interactions between roots, mycorrhizal fungi and PGPR can have synergistic effects on growth and systemic priming of wheat

An apoplastic peptide signal activates salicylic acid signalling in maize

Control of plant pathogen resistance or susceptibility largely depends on the promotion of either cell survival or cell death. In this context, papain-like cysteine proteases (PLCPs) regulate plant defence to drive cell death and protection against biotrophic pathogens. In maize (Zea mays), PLCPs are crucial in the orchestration of salicylic acid (SA)-dependent defence signalling. Despite this central role in immunity, it remains unknown how PLCPs are activated, and which downstream signals they induce to trigger plant immunity. Here, we present the discovery of an immune signalling peptide, Zea mays immune signalling peptide 1 (Zip1). A mass spectrometry approach identified the Zip1 peptide being produced after salicylic acid (SA) treatment. In vitro studies using recombinant proteins demonstrate that PLCPs are required to release bioactive Zip1 from its propeptide precursor (PROZIP1). Strikingly, Zip1 treatment strongly elicits SA accumulation in maize leaves. Moreover, RNAseq based transcriptome analyses revealed that Zip1 and SA treatments induce highly overlapping transcriptional changes. Consequently, Zip1 promotes the infection of the necrotrophic pathogen Botrytis cinerea in maize, while it reduces virulence of the biotrophic fungus Ustilago maydis. Together, Zip1 represents the previously missing signal that is released by PLCPs to activate SA defence signalling