A cytochrome P450 gene is differentially expressed in compatible and incompatible interactions between pepper (Capsicum annuum) and the anthracnose fungus, Colletotrichum gloeosporioides

The anthracnose fungus, Colletotrichum gloeosporioides, was previously shown to have an incompatible interaction with ripe-red fruit of pepper (Capsicum annuum). However, the fungus had a compatible interaction with unripe-mature-green fruit. Using mRNA differential display, we isolated and characterized a PepCYP gene expressed in the incompatible interaction. The PepCYP gene encodes a protein homologous to cytochrome P450 proteins containing a heme-binding domain. The expression level of PepCYP is higher in the incompatible interaction than in the compatible interaction, and then remains elevated in the incompatible interaction. In the compatible interaction, the expression of PepCYP is transient. The induction of PepCYP gene is up-regulated by wounding or jasmonic acid treatment during ripening. Analysis of PepCYP expression by in situ hybridization shows that the accumulation of PepCYP mRNA is localized in the epidermal cell layers, but not in the cortical cell layers. An examination of transverse sections of the fruits inoculated with the fungus shows that the fungus invades and colonizes the epidermal cell layers of the unripe fruit at 24 and 72 h after inoculation, respectively, but not those of the ripe fruit. These results suggest that the PepCYP gene product plays a role in the defense mechanism when the fungus invades and colonizes the epidermal cells of fruits in the incompatible interaction during the early fungal infection process.     

Differentially expressed proteins during an incompatible interaction between common bean and the fungus Pseudocercospora griseola

The common bean (Phaseolus vulgaris L.) is the main source of protein and an important source of minerals in several countries around the world. Angular leaf spot, caused by the fungus Pseudocercospora griseola, is one of the major diseases of the common bean. In this work, we used two-dimensional gel electrophoresis and mass spectrometry to analyze alterations in the proteome of common bean leaves challenged with an incompatible race of P. griseola. Twenty-three differentially expressed proteins were detected in leaves of cultivar AND 277 collected at 12, 24 and 48 h after inoculation. The proteins were digested with trypsin and submitted to MALDI-TOF/TOF and MicrOTOF-Q electrospray mass spectrometry. Nineteen of them were identified upon MS/MS fragmentation. Most of these proteins are involved with amino acid metabolism, terpenoid metabolism, phenylpropanoid biosynthesis, antioxidant systems, vitamin and cofactor metabolism, plant–pathogen interaction, carbohydrate metabolism, photosynthesis, or genetic information processing, showing that the interaction in this pathosystem affects different genes from various metabolic pathways and processes.     

Culture storage age and fungal re‐isolation from host‐tissue influence Colletotrichum spp. virulence to pepper fruits

Using resistant cultivars is the most sustainable and practical approach against plant diseases. Plant germplasm and breeding lines are selected and assayed against, usually, the most aggressive or virulent strains of a pathogen (e.g., fungus) that causes the disease. However, prolong storage of the pathogen in culture media could affect virulence that, consequently, also influence the outcome of the resistance assay. This study demonstrates that long‐term storage (at least a year) of Colletotrichum truncatum and C. scovillei, causal agents of pepper anthracnose, in potato dextrose agar (PDA) medium decreased the aggressiveness and virulence of the fungus in host‐pepper fruits. However, reintroduction of the pathogen to the host and isolation of the pathogen as the new inoculum, prior to inoculation assays, increased the virulence of the fungi. These findings suggest that re‐inoculation and re‐isolation of Colletotichum truncatum and C. scovillei that have been stored for at least 1 year in PDA medium are necessary when using fungal cultures in pathogenicity and plant resistance assays to achieve desirable, comparable and reliable results.     

菜豆炭疽菌有性态研究

在引起菜豆炭疽病的 Colletotrichum lindemuthianum (Sacc. Et Magn．)Br. EtCav．大量培养物中,发现一个在低温培养保存中丧失产生分生孢子能力而仅产生子囊壳的菌系。对其形态学、培养特征、单子囊孢子培养的研究及与有关种的形态比较结果表明,这个源于分生孢子的子囊菌培养物是菜豆小丛壳Glomerella lindemuthianum Shear。这个培养物经人工接种菜豆,再分离时首先产生无性态分生孢子,经数代培养又回复有性态,表明有性过程的产生是同宗配合的。这种有性与无性阶段之间的交替和联系,证明这是一个全型态真菌,即菜豆小丛壳 G. lindemuthianum 是无性态荣豆炭疽菌C．Lindemuthianum 的有性态。     

Beginning to understand the role of sugar carriers in Colletotrichum lindemuthianum: the function of the gene mfs1

Fungi of the Colletotrichum genus are among the most prominent phytopathogens that cause diseases with a considerable economic impact, such as anthracnose. The hemibiotrophic fungus Colletotrichum lindemuthianum (teleomorph Glomerella cingulata f. sp. phaseoli) is the causal agent of the anthracnose of the common bean; and similarly to other phytopathogens, it uses multiple strategies to gain access to different carbon sources from its host. In this study, we examine mfs1, a newly identified C. lindemuthianum hexose transporter. The mfs1 gene is expressed only during the necrotrophic phase of the fungus’ interaction within the plant and allows it to utilize the available sugars during this phase. The deletion of mfs1 gene resulted in differential growth of the fungus in a medium that contained glucose, mannose or fructose as the only carbon source. This study is the first to describe a hexose transporter in the hemibiotrophic pathogen C. lindemuthianum and to demonstrate the central role of this protein in capturing carbon sources during the necrotrophic development of the plant/pathogen interaction.     

Constitutive expression of a fungus-inducible carboxylesterase improves disease resistance in transgenic pepper plants

Main conclusion

Resistance against anthracnose fungi was enhanced in transgenic pepper plants that accumulated high levels of a carboxylesterase, PepEST in anthracnose-susceptible fruits, with a concurrent induction of antioxidant enzymes and SA-dependent PR proteins. A pepper esterase gene (PepEST) is highly expressed during the incompatible interaction between ripe fruits of pepper (Capsicum annuum L.) and a hemibiotrophic anthracnose fungus (Colletotrichum gloeosporioides). In this study, we found that exogenous application of recombinant PepEST protein on the surface of the unripe pepper fruits led to a potentiated state for disease resistance in the fruits, including generation of hydrogen peroxide and expression of pathogenesis-related (PR) genes that encode mostly small proteins with antimicrobial activity. To elucidate the role of PepEST in plant defense, we further developed transgenic pepper plants overexpressing PepEST under the control of CaMV 35S promoter. Molecular analysis confirmed the establishment of three independent transgenic lines carrying single copy of transgenes. The level of PepEST protein was estimated to be approximately 0.002 % of total soluble protein in transgenic fruits. In response to the anthracnose fungus, the transgenic fruits displayed higher expression of PR genes, PR3, PR5, PR10, and PepThi, than non-transgenic control fruits did. Moreover, immunolocalization results showed concurrent localization of ascorbate peroxidase (APX) and PR3 proteins, along with the PepEST protein, in the infected region of transgenic fruits. Disease rate analysis revealed significantly low occurrence of anthracnose disease in the transgenic fruits, approximately 30 % of that in non-transgenic fruits. Furthermore, the transgenic plants also exhibited resistance against C. acutatum and C. coccodes. Collectively, our results suggest that overexpression of PepEST in pepper confers enhanced resistance against the anthracnose fungi by activating the defense signaling pathways.

     

Wheat defense genes in fungal (Puccinia striiformis) infection

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most important diseases of wheat worldwide. To isolate defense-related genes against the pathogen, a suppression subtractive hybridization library was constructed for an incompatible interaction. From the library, 652 sequences were determined to be unigenes, of which 31 were determined as genes involved in signal transduction and 77 were predicted to encode defense-related proteins. Expression patterns of 12 selected signal transduction and defense-related genes were determined using quantitative real-time polymerase chain reaction. Signal transduction genes started increasing their expression at 12 h post inoculation (hpi), and expressions of the most of the transport and resistance-related genes were induced at 18 hpi. The gene expression results indicate specific molecular and cellular activities during the incompatible interaction between wheat and the stripe rust pathogen. In general, the expression increase of wheat signal transduction genes soon after inoculation with the pathogen inducing various defense-related genes, including reactive oxygen species, ATP-binding cassette (ABC) transporters, pathogenesis-related proteins, and genes involved in the phenylpropanoid pathway. The activities of these defense genes work in a sequential and concerted manner to result in a hypersensitive response.     

Ten rice peroxidases redundantly respond to multiple stresses including infection with rice blast fungus

Class III plant peroxidases are believed to function in diverse physiological processes including disease resistance and wound response, but predicted low substrate specificities and the presence of 70 or more isoforms have made it difficult to define a specific physiological function(s) for each gene. To select pathogen-responsive POX genes, we analyzed the expression profiles of 22 rice POX genes after infection with rice blast fungus. The expression of 10 POX genes among the 22 genes was induced after fungal inoculation in both compatible and incompatible hosts. Seven of the 10 POX genes were expressed at higher levels in the incompatible host than in the compatible host 6-24 h after inoculation by which time no fungus-induced lesions have appeared. Organ-specific expression and stress-induced expression by wounding and treatment with probenazole, an agrichemical against blast fungus, jasmonic acid, salicylic acid and 1-aminocyclopropane-1-carboxylate, a precursor of ethylene, indicated that rice POXs have individual characteristics and can be classified into several types. A comparison of the amino acid sequences of POXs showed that multiple isoforms with a high sequence similarity respond to stress in different or similar ways. Such redundant responses of POX genes may guarantee POX activities that are necessary for self-defense in plant tissues against environmental stresses including pathogen infection.     

Inhibition of fungal appressorium formation by pepper (Capsicum annuum) esterase

A pepper esterase gene (PepEST) that is highly expressed during an incompatible interaction between pepper (Capsicum annuum) and the anthracnose fungus Colletotrichum gloeosporioides has been previously cloned. Glutathione-S-transferase-tagged recombinant PepEST protein expressed in Escherichia coli showed substrate specificity for p-nitrophenyl esters. Inoculation of compatible unripe pepper fruits with C. gloeosporioides spores amended with the recombinant protein did not cause anthracnose symptoms on the fruit. The recombinant protein has no fungicidal activity, but it significantly inhibits appressorium formation of the anthracnose fungus in a dose-dependent manner. An esterase from porcine liver also inhibited appressorium formation, and the recombinant protein inhibited appressorium formation in the rice blast fungus, Magnaporthe grisea. Inhibition of appressorium formation in M. grisea by the recombinant protein was reversible by treatment with cyclic AMP (cAMP) or 1,16-hexadecanediol. The results suggest that the recombinant protein regulates appressorium formation by modulating the cAMP-dependent signaling pathway in this fungus. Taken together, the PepEST esterase activity can inhibit appressorium formation of C. gloeosporioides, which may result in protection of the unripe fruit against the fungus.     

Quantitative analysis of race-specific resistance to Colletotrichum lindemuthianum in common bean

Molecular genetic maps continue to play a major role in breeding of crop species. The common bean genetic map of the recombinant inbred line population IAC-UNA × CAL 143 (UC) has been used to detect loci controlling important agronomic traits in common bean. In the current study, new microsatellite markers were added to the UC map and the linkage analysis was refined using current genomic resources of common bean, in order to identify quantitative resistance loci (QRL) associated with different races of the anthracnose pathogen. A single race inoculation was conducted in greenhouse using four plants per plot. Both race-specific and joint-adjusted disease severity means, obtained from linear-mixed model, were used to perform multiple interval mapping (MIM) and multi-trait MIM (MTMIM). In total, 13 and 11 QRL were identified by MIM and MTMIM analyses, respectively; with nine being observed in both analyses. ANT02.1^UC and ANT07.1^UC showed major effects on resistance both for MIM and MTMIM. Common major QRL for resistance to the three anthracnose races were expected, since high genetic pairwise-correlation was observed between the race-specific and joint-adjusted disease severity means. Therewith, both ANT02.1 and ANT07.1 can be regarded as valuable targets for marker-assisted selection; and so, putative genes potentially involved in the resistance response were identified in these QRL regions. Minor effect QRL were also observed, showing differential affects either on race-specific or multi-trait analyses and may play a role on durable horizontal resistance. These results contribute to a better understanding of the host-pathogen interaction and to breeding for enhancing resistance to Colletotrichum lindemuthianum in common bean.