Functional analysis of the Alternaria brassicicola non-ribosomal peptide synthetase gene AbNPS2 reveals a role in conidial cell wall construction

Alternaria brassicicola is a necrotrophic pathogen causing black spot disease on virtually all cultivated Brassica crops worldwide. In many plant pathosystems fungal secondary metabolites derived from non-ribosomal peptide synthetases (NPSs) are phytotoxic virulence factors or are antibiotics thought to be important for niche competition with other micro-organisms. However, many of the functions of NPS genes and their products are largely unknown. In this study, we investigated the function of one of the A. brassicicola NPS genes, AbNPS2 . The predicted amino acid sequence of AbNPS2 showed high sequence similarity with A. brassicae , AbrePsy1, Cochliobolus heterostrophus , NPS4 and a Stagonospora nodorum NPS. The AbNPS2 open reading frame was predicted to be 22 kb in length and encodes a large protein (7195 amino acids) showing typical NPS modular organization. Gene expression analysis of AbNPS2 in wild-type fungus indicated that it is expressed almost exclusively in conidia and conidiophores, broadly in the reproductive developmental phase. AbNPS2 gene disruption mutants showed abnormal spore cell wall morphology and a decreased hydrophobicity phenotype. Conidia of abnps2 mutants displayed an aberrantly inflated cell wall and an increase in lipid bodies compared with wild-type. Further phenotypic analyses of abnps2 mutants showed decreased spore germination rates both in vitro and in vivo , and a marked reduction in sporulation in vivo compared with wild-type fungus. Moreover, virulence tests on Brassicas with abnps2 mutants revealed a significant reduction in lesion size compared with wild-type but only when aged spores were used in experiments. Collectively, these results indicate that AbNPS2 plays an important role in development and virulence.     

Profile and analysis of gene expression changes during early development in germinating spores of Ceratopteris richardii

Analysis of an expressed sequence tag library with more than 5,000 sequences from spores of the fern Ceratopteris richardii reveals that more than 3,900 of them represent distinct genes, and almost 70% of these have significant similarity to Arabidopsis (Arabidopsis thaliana) genes. Eight genes are common between three very different dormant plant systems, Ceratopteris spores, Arabidopsis seeds, and Arabidopsis pollen. We evaluated the pattern of mRNA abundance over the first 48 h of spore development using a microarray of cDNAs representing 3,207 distinct genes of C. richardii and determined the relative levels of RNA abundance for 3,143 of these genes using a Bayesian method of statistical analysis. More than 900 of them (29%) show a significant change between any of the five time points analyzed, and these have been annotated based on their sequence similarity with the Arabidopsis proteome. Novel data arising from these analyses identify genes likely to be critical for the germination and subsequent early development of diverse cells and tissues emerging from dormancy.     

Studies on the epidemiology of Alternaria brassicicola in Brassica oleracea seed production crops

Alternaria brassicicola lesions present on overwintered leaf litter of Brassica oleracea seed production crops produced high concentrations of spores in the spring, these were able to initiate new infections on foliage and subsequently on inflorescences and pods. A vertical disease gradient developed in maturing crops, the lowest pods becoming infected first and infection spreading slowly upwards. Spores were produced abundantly after 20 h leaf wetness at a mean temperature of 13°C or more. Their release was stimulated by a fall in relative humidity but inhibited at a constant high relative humidity resulting in a daily cycle in air spore concentrations with minimum numbers occurring in the early morning and maximum numbers in the early afternoon. For most of the growing season spore movement was restricted to within the crop, however, massive release of spores and subsequent distribution over a wide area occurred when the crop was cut and later threshed. Using semi-selective agar traps spores released at these times were detected up to 1800 m downwind of the parent crop and were instrumental in infecting nearby young crops destined for seed production in the following season.     

Requirement of functional ethylene-insensitive 2 gene for efficient resistance of Arabidopsis to infection by Botrytis cinerea

Inoculation of wild-type Arabidopsis plants with the fungus Alternaria brassicicola results in systemic induction of genes encoding a plant defensin (PDF1.2), a basic chitinase (PR-3), and an acidic hevein-like protein (PR-4). Pathogen-induced induction of these three genes is almost completely abolished in the ethylene-insensitive Arabidopsis mutant ein2-1. This indicates that a functional ethylene signal transduction component (EIN2) is required in this response. The ein2-1 mutants were found to be markedly more susceptible than wild-type plants to infection by two different strains of the gray mold fungus Botrytis cinerea. In contrast, no increased fungal colonization of ein2-1 mutants was observed after challenge with avirulent strains of either Peronospora parasitica or A. brassicicola. Our data support the conclusion that ethylene-controlled responses play a role in resistance of Arabidopsis to some but not all types of pathogens.     

The Glyoxylate Cycle in an Arbuscular Mycorrhizal Fungus. Carbon Flux and Gene Expression

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Lipid, which is the dominant form of stored carbon in the fungal partner and which fuels spore germination, is made by the fungus within the root and is exported to the extraradical mycelium. We tested the hypothesis that the glyoxylate cycle is central to the flow of carbon in the AM symbiosis. The results of (13)C labeling of germinating spores and extraradical mycelium with (13)C(2)-acetate and (13)C(2)-glycerol and analysis by nuclear magnetic resonance spectroscopy indicate that there are very substantial fluxes through the glyoxylate cycle in the fungal partner. Full-length sequences obtained by polymerase chain reaction from a cDNA library from germinating spores of the AM fungus Glomus intraradices showed strong homology to gene sequences for isocitrate lyase and malate synthase from plants and other fungal species. Quantitative real-time polymerase chain reaction measurements show that these genes are expressed at significant levels during the symbiosis. Glyoxysome-like bodies were observed by electron microscopy in fungal structures where the glyoxylate cycle is expected to be active, which is consistent with the presence in both enzyme sequences of motifs associated with glyoxysomal targeting. We also identified among several hundred expressed sequence tags several enzymes of primary metabolism whose expression during spore germination is consistent with previous labeling studies and with fluxes into and out of the glyoxylate cycle.     

Fungal spore attachment to substrata

A critical factor in the success of fungal growth is spore adhesion to host surfaces. Generating spores capable of rapid and firm bonding to their hosts is not only important for keeping spores from prematurely detaching from the host surface but can also serve as a trigger for spore germination and the development of infection structures. In this paper fungal spore adhesion mechanisms are reviewed as well as factors influencing spore adhesion, germination, and differentiation. This review ends with a brief discussion on the future of fungal adhesion research.     

Antifungal activity of venenatine, an indole alkaloid isolated fromAlstonia venenata

The indole alkaloid venenatine exhibited antifungal activity against some plant pathogenic and saprophytic fungi. Venenatine in an aqueous acetic acid solution inhibited spore germination of all the 10 tested fungi, Fusarium udum, Alternaria brassicicola, Ustilago cynodontis and Aspergillus flavus showed an especially high sensitivity towards this compound, exhibiting germination levels below 10%. The spore germination and colony development of the parasitic fungus Erysiphe pisi, which causes powdery mildew in pea (Pisum sativum), on excised leaves of pea was also significantly affected. Pre-inoculation rather than post inoculation treatment of the leaves was more inhibitory against spore germination and colony development.     

Characterization of the early response of Arabidopsis to Alternaria brassicicola infection using expression profiling

All tested accessions of Arabidopsis are resistant to the fungal pathogen Alternaria brassicicola. Resistance is compromised by pad3 or coi1 mutations, suggesting that it requires the Arabidopsis phytoalexin camalexin and jasmonic acid (JA)-dependent signaling, respectively. This contrasts with most well-studied Arabidopsis pathogens, which are controlled by salicylic acid-dependent responses and do not benefit from absence of camalexin or JA. Here, mutants with defects in camalexin synthesis (pad1, pad2, pad3, and pad5) or in JA signaling (pad1, coi1) were found to be more susceptible than wild type. Mutants with defects in salicylic acid (pad4 and sid2) or ethylene (ein2) signaling remained resistant. Plant responses to A. brassicicola were characterized using expression profiling. Plants showed dramatic gene expression changes within 12 h, persisting at 24 and 36 h. Wild-type and pad3 plants responded similarly, suggesting that pad3 does not have a major effect on signaling. The response of coi1 plants was quite different. Of the 645 genes induced by A. brassicicola in wild-type and pad3 plants, 265 required COI1 for full expression. It is likely that some of the COI1-dependent genes are important for resistance to A. brassicicola. Responses to A. brassicicola were compared with responses to Pseudomonas syringae infection. Despite the fact that these pathogens are limited by different defense responses, approximately 50% of the induced genes were induced in response to both pathogens. Among these, requirements for COI1 were consistent after infection by either pathogen, suggesting that the regulatory effect of COI1 is similar regardless of the initial stimulus.     

Real-time PCR monitoring of fungal development in Arabidopsis thaliana infected by Alternaria brassicicola and Botrytis cinerea.

Reliable methods for disease severity assessment are of crucial importance in the study of plant pathogen interactions, either for disease diagnostic on the field or to assess phenotypical differences in plants or pathogen strains. Currently, most of the assays used in fungal disease diagnostic rely on visual assessment of the symptoms, lesion diameter measurement or spore counting. However, these tests are tedious and often cannot discriminate between slightly different levels of resistance. Besides, they are not well suited to assess fungal development in the early phases of the infection, before macroscopical symptoms are visible or before sporulation. We describe here a pathogenicity assay based on the relative quantification of fungal and plant DNA in infected Arabidopsis thaliana leaves by means of real-time quantitative PCR. We show that it allows to monitor quantitatively the growth of the fungi Alternaria brassicicola and Botrytis cinerea in a sensitive and reliable way. Although highly sensitive, this test also exhibits a high robustness, which is crucial to significantly discriminate between lines displaying slightly different levels of resistance. Therefore, it allows to assess fungal development from the very first stages of infection and provides a fast and very practical alternative to currently described assays for phenotyping either plant mutant lines or fungal strains.     

The Fus3/Kss1 MAP kinase homolog Amk1 regulates the expression of genes encoding hydrolytic enzymes in Alternaria brassicicola

Mitogen-activated protein (MAP) kinases have been shown to be required for virulence in diverse phytopathogenic fungi. To study its role in pathogenicity, we disrupted the Amk1 MAP kinase gene, a homolog of the Fus3/Kss1 MAP kinases in Saccharomyces cerevisiae, in the necrotrophic Brassica pathogen, Alternaria brassicicola. The amk1 disruption mutants showed null pathogenicity on intact host plants. However, amk1 mutants were able to colonize host plants when they were inoculated on a physically damaged host surface, or when they were inoculated along with nutrient supplements. On intact plants, mutants expressed extremely low amounts of several hydrolytic enzyme genes that were induced over 10-fold in the wild-type during infection. These genes were also dramatically induced in the mutants on wounded plants. These results imply a correlation between virulence and the expression level of specific hydrolytic enzyme genes plus the presence of an unidentified pathway controlling these genes in addition to or in conjunction with the Amk1 pathway.     

Differential gene expression during teliospore germination in <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Ustilago maydis is a model fungal pathogen that induces the formation of tumors in maize. The tumor provides an environment for hyphal differentiation, leading to the formation of thick-walled, diploid teliospores. Such spores serve as a dispersal agent for smut and rust fungi, and their germination leads to new rounds of infection. The morphological changes that occur during teliospore germination in U. maydis have been described in detail. However, the specific molecular events that facilitate this process have not been identified. Through the construction and hybridization of microarrays containing a set of 3918 non-redundant cDNAs, we have identified genes that are differentially regulated during teliospore germination. Teliospores induced to germinate for 4 and 11 h were selected for comparison with dormant teliospores. Genes identified as differentially expressed included many that are presumably involved in as yet undescribed molecular events during teliospore germination, as well as characterized genes previously shown to be required for the process. This study represents the first large-scale investigation of changes in gene expression during teliospore germination.Electronic Supplementary Material Supplementary material is available for this article at