Epidermal chloroplasts are defense-related motile organelles equipped with plant immune components.

In addition to conspicuous large mesophyll chloroplasts, where most photosynthesis occurs, small epidermal chloroplasts have also been observed in plant leaves. However, the functional significance of this small organelle remains unclear. Here, we present evidence that Arabidopsis epidermal chloroplasts control the entry of fungal pathogens. In entry trials, specialized fungal cells called appressoria triggered dynamic movement of epidermal chloroplasts. This movement is controlled by common regulators of mesophyll chloroplast photorelocation movement, designated as the epidermal chloroplast response (ECR). The ECR occurs when the PEN2 myrosinase-related higher-layer antifungal system becomes ineffective, and blockage of the distinct steps of the ECR commonly decreases preinvasive nonhost resistance against fungi. Furthermore, immune components were preferentially localized to epidermal chloroplasts, contributing to antifungal nonhost resistance in the pen2 background. Our findings reveal that atypical small chloroplasts act as defense-related motile organelles by specifically positioning immune components in the plant epidermis, which is the first site of contact between the plant and pathogens. Thus, this work deepens our understanding of the functions of epidermal chloroplasts.

A novel effector CfEC92 of Colletotrichum fructicola contributes to glomerella leaf spot virulence by suppressing plant defences at the early infection phase.

The ascomycete fungus Colletotrichum fructicola causes diseases on a broad range of plant species. On susceptible cultivars of apple, it induces severe early defoliation and fruit spots, named glomerella leaf spot (GLS), but the mechanisms of pathogenicity have remained elusive. Phytopathogens exhibit small secreted effectors to advance host infection by manipulating host immune reactions. We report the identification and characterization of CfEC92, an effector required for C. fructicola virulence. CfEC92 is a Colletotrichum-specific small secreted protein that suppresses BAX-triggered cell death in Nicotiana benthamiana. Accumulation of the gene transcript was barely detectable in conidia or vegetative hyphae, but was highly up-regulated in appressoria formed during early apple leaf infection. Gene deletion mutants were not affected in vegetative growth, appressorium formation, or appressorium-mediated cellophane penetration. However, the mutants were significantly reduced in virulence toward apple leaves and fruits. Microscopic examination indicated that infection by the deletion mutants elicited elevated deposition of papillae at the penetration sites, and formation of infection vesicles and primary hyphae was retarded. Signal peptide activity, subcellular localization, and cell death-suppressive activity (without signal peptide) assays suggest that CfEC92 could be secreted and perform virulence functions inside plant cells. RNA sequencing and quantitative reverse transcription PCR results confirmed that the deletion mutants triggered elevated host defence reactions. Our results strongly support the interpretation that CfEC92 contributes to C. fructicola virulence as a plant immunity suppressor at the early infection phase.

Small RNA-Based Antiviral Defense in the Phytopathogenic Fungus Colletotrichum higginsianum.

Even though the fungal kingdom contains more than 3 million species, little is known about the biological roles of RNA silencing in fungi. The Colletotrichum genus comprises fungal species that are pathogenic for a wide range of crop species worldwide. To investigate the role of RNA silencing in the ascomycete fungus Colletotrichum higginsianum, knock-out mutants affecting genes for three RNA-dependent RNA polymerase (RDR), two Dicer-like (DCL), and two Argonaute (AGO) proteins were generated by targeted gene replacement. No effects were observed on vegetative growth for any mutant strain when grown on complex or minimal media. However, Δdcl1, Δdcl1Δdcl2 double mutant, and Δago1 strains showed severe defects in conidiation and conidia morphology. Total RNA transcripts and small RNA populations were analyzed in parental and mutant strains. The greatest effects on both RNA populations was observed in the Δdcl1, Δdcl1Δdcl2, and Δago1 strains, in which a previously uncharacterized dsRNA mycovirus [termed Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1)] was derepressed. Phylogenetic analyses clearly showed a close relationship between ChNRV1 and members of the segmented Partitiviridae family, despite the non-segmented nature of the genome. Immunoprecipitation of small RNAs associated with AGO1 showed abundant loading of 5'U-containing viral siRNA. C. higginsianum parental and Δdcl1 mutant strains cured of ChNRV1 revealed that the conidiation and spore morphology defects were primarily caused by ChNRV1. Based on these results, RNA silencing involving ChDCL1 and ChAGO1 in C. higginsianum is proposed to function as an antiviral mechanism.

Attenuation of PAMP-triggered immunity in maize requires down-regulation of the key ß-1,6-glucan synthesis genes KRE5 and KRE6 in biotrophic hyphae of Colletotrichum graminicola.

In plants, pathogen defense is initiated by recognition of pathogen-associated molecular patterns (PAMPs) via plasma membrane-localized pattern-recognition receptors (PRRs). Fungal structural cell wall polymers such as branched ß-glucans are essential for infection structure rigidity and pathogenicity, but at the same time represent PAMPs. Kre5 and Kre6 are key enzymes in ß-1,6-glucan synthesis and formation of branch points of the ß-glucan network. In spite of the importance of branched ß-glucan for hyphal rigidity and plant-fungus interactions, neither the role of KRE5 and KRE6 in pathogenesis nor mechanisms allowing circumventing branched ß-glucan-triggered immune responses are known. We functionally characterized KRE5 and KRE6 of the ascomycete Colletotrichum graminicola, a hemibiotroph that infects maize (Zea mays). After appressorial plant invasion, this fungus sequentially differentiates biotrophic and highly destructive necrotrophic hyphae. RNAi-mediated reduction of KRE5 and KRE6 transcript abundance caused appressoria to burst and swelling of necrotrophic hyphae, indicating that ß-1,6-glucosidic bonds are essential in these cells. Live cell imaging employing KRE5:mCherry and KRE6:mCherry knock-in strains and probing of infection structures with a YFP-conjugated ß-1,6-glucan-binding protein showed expression of these genes and exposure of ß-1,6-glucan in conidia, appressoria and necrotrophic, but not in biotrophic hyphae. Overexpression of KRE5 and KRE6 in biotrophic hyphae led to activation of broad-spectrum plant defense responses, including papilla and H2 O2 formation, as well as transcriptional activation of several defense-related genes. Collectively, our results strongly suggest that down-regulation of synthesis and avoidance of exposure of branched ß-1,3-ß-1,6-glucan in biotrophic hyphae is required for attenuation of plant immune responses.

Danger peptide receptor signaling in plants ensures basal immunity upon pathogen-induced depletion of BAK1.

Pathogens infect a host by suppressing defense responses induced upon recognition of microbe-associated molecular patterns (MAMPs). Despite this suppression, MAMP receptors mediate basal resistance to limit host susceptibility, via a process that is poorly understood. The Arabidopsis leucine-rich repeat (LRR) receptor kinase BAK1 associates and functions with different cell surface LRR receptors for a wide range of ligands, including MAMPs. We report that BAK1 depletion is linked to defense activation through the endogenous PROPEP peptides (Pep epitopes) and their LRR receptor kinases PEPR1/PEPR2, despite critical defects in MAMP signaling. In bak1-knockout plants, PEPR elicitation results in extensive cell death and the prioritization of salicylate-based defenses over jasmonate-based defenses, in addition to elevated proligand and receptor accumulation. BAK1 disruption stimulates the release of PROPEP3, produced in response to Pep application and during pathogen challenge, and renders PEPRs necessary for basal resistance. These findings are biologically relevant, since specific BAK1 depletion coincides with PEPR-dependent resistance to the fungal pathogen Colletotrichum higginsianum. Thus, the PEPR pathway ensures basal resistance when MAMP-triggered defenses are compromised by BAK1 depletion.

Identification and molecular mapping of a wheat gene for resistance to an unadapted isolate of Colletotrichum cereale.

To elucidate genetic mechanisms of host species specificity between graminicolous anthracnose fungi and gramineous plants, infection assays were performed with a Sorghum isolate (Colletotrichum sublineolum), a Digitaria isolate (C. hanaui), a Polypogon isolate (C. cereale), and an Avena isolate (C. cereale). They were specifically virulent on the plants from which they were isolated. When 72 wheat lines were inoculated with an unadapted isolate from Asia Minor bluegrass (Cgp29), however, some exceptional cultivars were recognized. Although most cultivars were resistant to Cgp29, 'Hope' was susceptible. In F2 populations derived from crosses between three resistant cultivars-'Norin 4' (N4), 'Chinese Spring' (CS), and 'Shin-chunaga' (Sch)-and the susceptible Hope, resistant and susceptible seedlings segregated in a 3:1 ratio, suggesting that a major gene is involved in the resistance of each cultivar to Cgp29. In F2 populations derived from crosses between the three resistant cultivars, all seedlings were resistant, suggesting that these three cultivars carry the same gene. This resistance gene was designated as "resistance to Colletotrichum cereale 1" (Rcc1). Analysis with the CS-Hope chromosome substitution lines and molecular mapping revealed that Rcc1 was located on the long arm of chromosome 5A. Cytologically, Rcc1 was mainly associated with hypersensitive reaction. These results suggest that major genes similar to those controlling cultivar specificity are involved in the resistance of wheat against the unadapted isolate of C. cereale.

Glutathione and tryptophan metabolism are required for Arabidopsis immunity during the hypersensitive response to hemibiotrophs.

The hypersensitive response (HR) is a type of strong immune response found in plants that is accompanied by localized cell death. However, it is unclear how HR can block a broad range of pathogens with different infective modes. In this study, we report that γ-glutamylcysteine synthetase GSH1, which is critical for glutathione biosynthesis, and tryptophan (Trp) metabolism contribute to HR and block development of fungal pathogens with hemibiotrophic infective modes. We found that GSH1 is involved in the penetration2 (PEN2)-based entry control of the nonadapted hemibiotroph Colletotrichum gloeosporioides. However, Arabidopsis mutants specifically defective in entry control terminated further growth of the pathogen in the presence of HR cell death, whereas gsh1 mutants supported pathogen invasive growth in planta, demonstrating the requirement of GSH1 for postinvasive nonhost resistance. Remarkably, on the basis of the phenotypic and metabolic analysis of Arabidopsis mutants defective in Trp metabolism, we showed that biosynthesis of Trp-derived phytochemicals is also essential for resistance to C. gloeosporioides during postinvasive HR. By contrast, GSH1 and these metabolites are likely to be dispensable for the induction of cell death during postinvasive HR. Furthermore, the resistance to Ralstonia solanacearum 1/resistance to Pseudomonas syringae 4 dual Resistance gene-dependent immunity of Arabidopsis to the adapted hemibiotroph shared GSH1 and cytochromes P450 CYP79B2/CYP79B3 with postinvasive nonhost resistance, whereas resistance to P. syringae pv. maculicola 1 and resistance to P. syringae 2-based Resistance gene resistance against bacterial pathogens did not. These data suggest that the synthesis of glutathione and Trp-derived metabolites during HR play crucial roles in terminating the invasive growth of both nonadapted and adapted hemibiotrophs.

Determination of the variations in levels of phenolic compounds in soybean (Glycine max Merr.) sprouts infected by anthracnose (Colletotrichum gloeosporioides).

BACKGROUND: Soybean sprouts (Kongnamool) are one of the most popular and nutritive traditional vegetables in East Asia. Anthracnose caused by Colletotrichum gloeosporioides is one of the most serious diseases of soybean sprouts. In order to obtain basic information for breeding and/or selecting soybean genotypes with increased natural defense against anthracnose, phenolic compounds were profiled for healthy and infected soybean (Glycine max Merr.) sprouts by using high-performance liquid chromatography coupled with tandem mass spectrometry. RESULTS: Tryptophan and eight phenolic compounds (daidzin, genistin, malonyldaidzin, malonylgenistin, daidzein, glycitein, genistein and coumestrol) were determined from healthy and inoculated sprouts. Total identified phenolic content was 40.02 ± 0.03 mg kg⁻¹, 99.4% of which was isoflavones. CONCLUSION: The monitoring suggested that de novo induced glycitein appeared to act as a phytoalexin in the defence mechanism of the soybean sprouts against C. gloeosporioides, and constitutively formed seven phenolic components that functioned as phytoanticipins in the diseased soybean sprouts.

Isolation of a strawberry gene fragment encoding an actin depolymerizing factor-like protein from genotypes resistant to Colletotrichum acutatum.

Actin depolymerizing factors (ADFs) have been recently implicated in plant defense against pathogenic fungi, associated with the cytoskeletal rearrangements that contribute to establish an effective barrier against fungal ingress. In this work, we identified a DNA fragment corresponding to a part of a gene predicted to encode an ADF-like protein in genotypes of Fragaria ananassa resistant to the fungus Colletotrichum acutatum. Bulked segregant analysis combined with AFLP was used to identify polymorphisms linked to resistance in hybrids derived from the cross between the resistant cultivar 'Sweet Charlie' and the susceptible cultivar 'Pájaro'. The sequence of one out of three polymorphic bands detected showed significant BLASTX hits to ADF proteins from other plants. Two possible exons were identified and bioinformatic analysis revealed the presence of the ADF homology domain with two actin-binding sites, an N-terminal phosphorylation site, and a nuclear localization signal. In addition to its possible application in strawberry breeding programs, these finding may contribute to investigate the role of ADFs in plant resistance against fungi.

Protocolo para la formación de microtubérculos de ñame (Dioscorea alata L) en sistema de inmersión temporal / A protocol for yam (Dioscorea alata L) microtuber formation in temporary immersion system

Con el propósito de desarrollar un protocolo para la formación de microtubérculos en el clon de ñame Pacala Duclos (Dioscorea alata L) en sistema de inmersión temporal, que pudiera ser usado como alternativa para la propagación de esta especie, se deinieron como objetivos de trabajo determinar el efecto del tiempo y la frecuencia de inmersión, así como la inluencia del volumen de medio de cultivo por planta cultivada in vitro. Con el empleo de 15 min de inmersión y una frecuencia de inmersión cada 6 horas, se alcanzaron los mejores resultados en cuanto al número de microtubérculos formados por planta. Con este tiempo y frecuencia de inmersión los microtubérculos presentaron la mayor masa fresca y seca, así como el mayor diámetro. Además, a las 18 semanas de cultivo se obtuvo el mayor número total de microtubérculos por sistema y el mayor número de microtubérculos aprovechables como material vegetal de propagación. En cuanto al volumen de medio de cultivo por planta, con 60 ml de medio de cultivo por planta in vitro se alcanzó el mayor número de microtubérculos aprovechables, los cuales presentaron el contenido más alto de materia seca. Los microtubérculos obtenidos en este tipo de sistema de inmersión temporal presentaron una masa fresca superior a 2,40 gMF, lo cual podría permitir su uso como material de plantación directo a campo

The following working objectives were deined for developing a protocol for ´Pacala Duclos´ yam clone (Dioscorea alata L) microtuber formation in temporary immersion systems (TIS): time effect, immersion frequency and culture medium volume inluence per in vitro cultivated plant. The results led to deining that the highest performance regarding microtuber number formed per plant was achieved with a 15-minute immersion time and six-hourly immersion frequency; the microtubers presented the greatest dry and fresh weight and largest diameter at such immersion time and frequency. Furthermore, the highest total number of microtubers per system and the largest number of microtubers usable for planting (vegetal propagation) material were obtained after 18 weeks’ culture. Regarding culture medium volume per plant, the greatest amount of usable microtubers was achieved with 60 ml per in vitro plant, microtubers presenting the highest dry matter content. As microtubers obtained in this type of TIS had fresh weights higher than 2.40 gFW they could be used as direct planting material in ield conditions

Alfalfa benefits from Medicago truncatula: the RCT1 gene from M. truncatula confers broad-spectrum resistance to anthracnose in alfalfa.

Alfalfa is economically the most important forage legume worldwide. A recurrent challenge to alfalfa production is the significant yield loss caused by disease. Although knowledge of molecular mechanisms underlying host resistance should facilitate the genetic improvement of alfalfa, the acquisition of such knowledge is hampered by alfalfa's tetrasomic inheritance and outcrossing nature. However, alfalfa is congeneric with the reference legume Medicago truncatula, providing an opportunity to use M. truncatula as a surrogate to clone the counterparts of many agronomically important genes in alfalfa. In particular, the high degree of sequence identity and remarkably conserved genome structure and function between the two species enables M. truncatula genes to be used directly in alfalfa improvement. Here we report the map-based cloning of RCT1, a host resistance (R) gene in M. truncatula that confers resistance to multiple races of Colletotrichum trifolii, a hemibiotrophic fungal pathogen that causes anthracnose disease of alfalfa. RCT1 is a member of the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class of plant R genes and confers broad-spectrum anthracnose resistance when transferred into susceptible alfalfa plants. Thus, RCT1 provides a novel resource to develop anthracnose-resistant alfalfa cultivars and contributes to our understanding of host resistance against the fungal genus Colletotrichum. This work demonstrates the potential of using M. truncatula genes for genetic improvement of alfalfa.

Colutellin A, an immunosuppressive peptide from Colletotrichum dematium.

Colletotrichum dematium is an endophytic fungus recovered from a Pteromischum sp. growing in a tropical forest in Costa Rica. This fungus makes a novel peptide antimycotic, colutellin A, with a MIC of 3.6 microg ml(-1) (48 h) against Botrytis cinerea and Sclerotinia sclerotiorum. Collutellin A has a mass of 1127.7 Da and contains residues of Ile, Val, Ser, N-methyl-Val and beta-aminoisobutryic acid in nominal molar ratios of 3 : 2 : 1 : 1 : 1, respectively. Independent lines of evidence suggest that the peptide is cyclic and sequences of Val-Ile-Ser-Ile and Ile-Pro-Val have been deduced by MS/MS as well as Edman degradation methods. Colutellin A inhibited CD4(+) T-cell activation of interleukin 2 (IL-2) production with an IC(50) of 167.3+/-0.38 nM, whereas cyclosporin A in the same test yielded a value of 61.8 nM. Inhibition of IL-2 production by collutellin A at such a low concentration indicates the potential immunosuppressive activity of this compound. In repeated experiments, cyclosporin A at or above 8 microg ml(-1) exhibited high levels of cytotoxicity on human peripheral blood mononuclear cells, whereas collutellin A or DMSO (carrier) alone, after 24 and 48 h of culture, exhibited no toxicity. Because of these properties collutellin A has potential as a novel immunosuppressive drug.

Triterpene phytoalexins from strawberry fruit.

Strawberry cv. Houkouwase is resistant to infection by Colletotrichum fragariae. The formation of antifungal compounds was observed in unripe fruit which had been wounded and inoculated with conidia of C. musae. Three antifungal compounds were isolated and identified as euscaphic acid, tormentic acid and myrianthic acid. Myrianthic acid inhibited the growth of C. musae at 3 microg, and euscaphic and tormentic acids showed inhibitory effects at 100 microg. A quantitative analysis of their contents showed that the triterpenes increased in wounded fruit, and in wounded and inoculated fruit, but not in non-treated fruit. These findings indicate that unripe fruit of Houkouwase produced the triterpenes as phytoalexins. The triterpene phytoalexins seem to be involved in the resistance of strawberry to the fungus.

Immunomagnetic purification of Colletotrichum lindemuthianum appressoria.

We developed a method to purify appressoria of the bean anthracnose fungus Colletotrichum lindemuthianum for biochemical analysis of the cell surface and to compare appressoria with other fungal structures. We used immunomagnetic separation after incubation of infected bean leaf homogenates with a monoclonal antibody that binds strongly to the appressoria. Preparations with a purity of >90% could be obtained. Examination of the purified appressoria by transmission electron microscopy showed that most had lost their cytoplasm. However, the plasma membrane was retained, suggesting that there is some form of attachment of this membrane to the cell wall. The purified appressoria can be used for studies of their cell surface, and we have shown that there are clear differences in the glycoprotein constituents of cell walls of appressoria compared with mycelium.

Spore surface glycoproteins of Colletotrichum lindemuthianum are recognized by a monoclonal antibody which inhibits adhesion to polystyrene.

Conidia (spores) of Colletotrichum lindemuthianum, a fungal plant pathogen causing bean anthracnose, adhere to the aerial parts of host plants to initiate the infection process. These spores possess a fibrillar 'spore coat' as well as a cell wall. In a previous study a mAb, UB20, was raised that recognized glycoproteins on the spore surface. In this study UB20 was used to localize and characterize these glycoproteins and to investigate their possible role in adhesion. Glycoproteins recognized by UB20 were concentrated on the outer surface of the spore coat and, to a lesser extent, at the plasma membrane/cell wall interface. Extraction of spores with hot water or 0.2% SDS resulted in removal of the spore coat. Western blotting with UB20 showed that a relatively small number of glycoproteins were extracted by these procedures, including a major component at 110 kDa. Biotinylation of carbohydrate moieties, together with cell fractionation, confirmed that these glycoproteins were exposed at the surface of the spores. In adhesion assays, > 90% of ungerminated conidia attached to polystyrene Petri dishes within 30 min. UB20 IgG at low concentrations inhibited attachment in an antigen-specific manner. This suggests that the glycoproteins recognized by this mAb may function in the initial rapid attachment of conidia to hydrophobic substrata. Polystyrene microspheres bound selectively to the 110 kDa glycoprotein in Western blots, providing further evidence that this component could mediate interactions with hydrophobic substrata.