Two genes in a pathogenicity gene cluster encoding secreted proteins are required for appressorial penetration and infection of the maize anthracnose fungus Colletotrichum graminicola.

To avoid pathogen-associated molecular pattern recognition, the hemibiotrophic maize pathogen Colletotrichum graminicola secretes proteins mediating the establishment of biotrophy. Targeted deletion of 26 individual candidate genes and seven gene clusters comprising 32 genes of C. graminicola identified a pathogenicity cluster (CLU5) of five co-linear genes, all of which, with the exception of CLU5b, encode secreted proteins. Targeted deletion of all genes of CLU5 revealed that CLU5a and CLU5d are required for full appressorial penetration competence, with virulence deficiencies independent of the host genotype and organ inoculated. Cytorrhysis experiments and microscopy showed that Δclu5a mutants form pressurized appressoria, but they are hampered in forming penetration pores and fail to differentiate a penetration peg. Whereas Δclu5d mutants elicited WT-like papillae, albeit at increased frequencies, papillae induced by Δclu5a mutants were much smaller than those elicited by the WT. Synteny of CLU5 is not only conserved in Colletotrichum spp. but also in additional species of Sordariomycetes including insect pathogens and saprophytes suggesting importance of CLU5 for fungal biology. Since CLU5a and CLU5d also occur in non-pathogenic fungi and since they are expressed prior to plant invasion and even in vegetative hyphae, the encoded proteins probably do not act primarily as effectors.

Correspondence between symptom development of Colletotrichum graminicola and fungal biomass, quantified by a newly developed qPCR assay, depends on the maize variety.

BACKGROUND: Penetration attempts of the hemibiotroph Colletotrichum graminicola may activate PAMP-triggered immunity (PTI) on different cultivars of Zea mays to different extent. However, in most events, this does not prevent the establishment of a compatible pathogenic interaction. In this study, we investigate the extent to which the host variety influences PTI. Furthermore, we assess whether visual disease symptoms occurring on different maize varieties reliably reflect fungal biomass development in planta as determined by qPCR and GFP tracing. RESULTS: Employing a set of four maize varieties, which were selected from a panel of 27 varieties, for in-depth assessment of pathogenesis of the wild type strain of C. graminicola, revealed considerable differences in susceptibility as evidenced by symptom severity that decreased from variety Golden Jubilee to Mikado to Farmtop to B73. However, a newly developed qPCR assay and microscopical observation of a GFP-labelled strain showed that disease symptoms are in some instances inconsistent when compared with other indicators of susceptibility. Of the four varieties assessed, either Golden Jubilee, Mikado and B73, or Golden Jubilee, Farmtop and B73 showed a direct correlation between symptom and fungal biomass development. In a pairwise comparison, however, Mikado and Farmtop showed an inverse correlation for these features. CONCLUSIONS: The genotype of maize contributes to the severity of symptoms resulting from an infection with C. graminicola. Partially, this may be attributed to the extent of PTI activated in different varieties, as reflected by papilla formation. Furthermore, when evaluating the susceptibility of a variety, it should be considered that symptom severity must not have to reflect the extent of fungal growth in the infected tissue.

Compositions of fungal secretomes indicate a greater impact of phylogenetic history than lifestyle adaptation.

BACKGROUND: Since the first fungal genome sequences became available, investigators have been employing comparative genomics to understand how fungi have evolved to occupy diverse ecological niches. The secretome, i.e. the entirety of all proteins secreted by an organism, is of particular importance, as by these proteins fungi acquire nutrients and communicate with their surroundings. RESULTS: It is generally assumed that fungi with similar nutritional lifestyles have similar secretome compositions. In this study, we test this hypothesis by annotating and comparing the soluble secretomes, defined as the sets of proteins containing classical signal peptides but lacking transmembrane domains of fungi representing a broad diversity of nutritional lifestyles. Secretome size correlates with phylogeny and to a lesser extent with lifestyle. Plant pathogens and saprophytes have larger secretomes than animal pathogens. Small secreted cysteine-rich proteins (SSCPs), which may comprise many effectors important for the interaction of plant pathogens with their hosts, are defined here to have a mature length of ≤ 300 aa residues, at least four cysteines, and a total cysteine content of ≥5%. SSCPs are found enriched in the secretomes of the Pezizomycotina and Basidiomycota in comparison to Saccharomycotina. Relative SSCP content is noticeably higher in plant pathogens than in animal pathogens, while saprophytes were in between and closer to plant pathogens. Expansions and contractions of gene families and in the number of occurrences of functional domains are largely lineage specific, e.g. contraction of glycoside hydrolases in Saccharomycotina, and are only weakly correlated with lifestyle. However, within a given lifestyle a few general trends exist, such as the expansion of secreted family M14 metallopeptidases and chitin-binding proteins in plant pathogenic Pezizomycotina. CONCLUSIONS: While the secretomes of fungi with similar lifestyles share certain characteristics, the expansion and contraction of gene families is largely lineage specific, and not shared among all fungi of a given lifestyle.

A novel, lactase-based selection and strain improvement strategy for recombinant protein expression in Kluyveromyces lactis.

BACKGROUND: The Crabtree-negative yeast species Kluyveromyces lactis has been established as an attractive microbial expression system for recombinant proteins at industrial scale. Its LAC genes allow for utilization of the inexpensive sugar lactose as a sole source of carbon and energy. Lactose efficiently induces the LAC4 promoter, which can be used to drive regulated expression of heterologous genes. So far, strain manipulation of K. lactis by homologous recombination was hampered by the high rate of non-homologous end-joining. RESULTS: Selection for growth on lactose was applied to target the insertion of heterologous genes downstream of the LAC4 promoter into the K. lactis genome and found to yield high numbers of positive transformants. Concurrent reconstitution of the ß-galactosidase gene indicated the desired integration event of the expression cassette, and ß-galactosidase activity measurements were used to monitor gene expression for strain improvement and fermentation optimization. The system was particularly improved by usage of a cell lysis resistant strain, VAK367-D4, which allowed for protein accumulation in long-term fermentation. Further optimization was achieved by increased gene dosage of KlGAL4 encoding the activator of lactose and galactose metabolic genes that led to elevated transcription rates. Pilot experiments were performed with strains expressing a single-chain antibody fragment (scFvox) and a viral envelope protein (BVDV-E2), respectively. scFvox was shown to be secreted into the culture medium in an active, epitope-binding form indicating correct processing and protein folding; the E2 protein could be expressed intracellularly. Further data on the influence of protein toxicity on batch fermentation and potential post-transcriptional bottlenecks in protein accumulation were obtained. CONCLUSIONS: A novel Kluyveromyces lactis host-vector system was developed that places heterologous genes under the control of the chromosomal LAC4 promoter and that allows monitoring of its transcription rates by ß-galactosidase measurement. The procedure is rapid and efficient, and the resulting recombinant strains contain no foreign genes other than the gene of interest. The recombinant strains can be grown non-selectively in rich medium and stably maintained even when the gene product exerts protein toxicity.

The yeast signal sequence trap identifies secreted proteins of the hemibiotrophic corn pathogen Colletotrichum graminicola.

The hemibiotroph Colletotrichum graminicola is the causal agent of stem rot and leaf anthracnose on Zea mays. Following penetration of epidermal cells, the fungus enters a short biotrophic phase, followed by a destructive necrotrophic phase of pathogenesis. During both phases, secreted fungal proteins are supposed to determine progress and success of the infection. To identify genes encoding such proteins, we constructed a yeast signal sequence trap (YSST) cDNA-library from RNA extracted from mycelium grown in vitro on corn cell walls and leaf extract. Of the 103 identified unigenes, 50 showed significant similarities to genes with a reported function, 25 sequences were similar to genes without a known function, and 28 sequences showed no similarity to entries in the databases. Macroarray hybridization and quantitative reverse-transcriptase polymerase chain reaction confirmed that most genes identified by the YSST screen are expressed in planta. Other than some genes that were constantly expressed, a larger set showed peaks of transcript abundances at specific phases of pathogenesis. Another set exhibited biphasic expression with peaks at the biotrophic and necrotrophic phase. Transcript analyses of in vitro-grown cultures revealed that several of the genes identified by the YSST screen were induced by the addition of corn leaf components, indicating that host-derived factors may have mimicked the host milieu.

Divergent Evolution of the Transcriptional Network Controlled by Snf1-Interacting Protein Sip4 in Budding Yeasts.

Cellular responses to starvation are of ancient origin since nutrient limitation has always been a common challenge to the stability of living systems. Hence, signaling molecules involved in sensing or transducing information about limiting metabolites are highly conserved, whereas transcription factors and the genes they regulate have diverged. In eukaryotes the AMP-activated protein kinase (AMPK) functions as a central regulator of cellular energy homeostasis. The yeast AMPK ortholog SNF1 controls the transcriptional network that counteracts carbon starvation conditions by regulating a set of transcription factors. Among those Cat8 and Sip4 have overlapping DNA-binding specificity for so-called carbon source responsive elements and induce target genes upon SNF1 activation. To analyze the evolution of the Cat8-Sip4 controlled transcriptional network we have compared the response to carbon limitation of Saccharomyces cerevisiae to that of Kluyveromyces lactis. In high glucose, S. cerevisiae displays tumor cell-like aerobic fermentation and repression of respiration (Crabtree-positive) while K. lactis has a respiratory-fermentative life-style, respiration being regulated by oxygen availability (Crabtree-negative), which is typical for many yeasts and for differentiated higher cells. We demonstrate divergent evolution of the Cat8-Sip4 network and present evidence that a role of Sip4 in controlling anabolic metabolism has been lost in the Saccharomyces lineage. We find that in K. lactis, but not in S. cerevisiae, the Sip4 protein plays an essential role in C2 carbon assimilation including induction of the glyoxylate cycle and the carnitine shuttle genes. Induction of KlSIP4 gene expression by KlCat8 is essential under these growth conditions and a primary function of KlCat8. Both KlCat8 and KlSip4 are involved in the regulation of lactose metabolism in K. lactis. In chromatin-immunoprecipitation experiments we demonstrate binding of both, KlSip4 and KlCat8, to selected CSREs and provide evidence that KlSip4 counteracts KlCat8-mediated transcription activation by competing for binding to some but not all CSREs. The finding that the hierarchical relationship of these transcription factors differs between K. lactis and S. cerevisiae and that the sets of target genes have diverged contributes to explaining the phenotypic differences in metabolic life-style.

Evolution of gene families: the multidrug resistance transporter genes in five related yeast species.

The available genomic sequences of five closely related hemiascomycetous yeast species (Kluyveromyces lactis, Kluyveromyces waltii, Candida glabrata, Ashbya (Eremothecium) gossypii with Saccharomyces cerevisiae as a reference) were analysed to identify multidrug resistance (MDR) transport proteins belonging to the ATP-binding cassette (ABC) and major facilitator superfamilies (MFS), respectively. The phylogenetic trees clearly demonstrate that a similar set of gene (sub)families already existed in the common ancestor of all five fungal species studied. However, striking differences exist between the two superfamilies with respect to the evolution of the various subfamilies. Within the ABC superfamily all six half-size transporters with six transmembrane-spanning domains (TMs) and most full-size transporters with 12 TMs have one and only one gene per genome. An exception is the PDR family, in which gene duplications and deletions have occurred independently in individual genomes. Among the MFS transporters, the DHA2 family (TC 2.A.1.3) is more variable between species than the DHA1 family (TC 2.A.1.2). Conserved gene order relationships allow to trace the evolution of most (sub)families, for which the Kluyveromyces lactis genome can serve as an optimal scaffold. Cross-species sequence alignment of orthologous upstream gene sequences led to the identification of conserved sequence motifs ("phylogenetic footprints"). Almost half of them match known sequence motifs for the MDR regulators described in S. cerevisiae. The biological significance of those and of the novel predicted motifs awaits to be confirmed experimentally.