Transcriptomic Response of Fusarium verticillioides to Variably Inhibitory Environmental Isolates of Streptomyces.

Fusarium verticillioides is a mycotoxigenic fungus that is a threat to food and feed safety due to its common infection of maize, a global staple crop. A proposed strategy to combat this threat is the use of biological control bacteria that can inhibit the fungus and reduce mycotoxin contamination. In this study, the effect of multiple environmental isolates of Streptomyces on F. verticillioides was examined via transcriptome analysis. The Streptomyces strains ranged from inducing no visible response to dramatic growth inhibition. Transcriptionally, F. verticillioides responded proportionally to strain inhibition with either little to no transcript changes to thousands of genes being differentially expressed. Expression changes in multiple F. verticillioides putative secondary metabolite gene clusters was observed. Interestingly, genes involved in the fusaric acid gene cluster were suppressed by inhibitory strains of Streptomyces. A F. verticillioides beta-lactamase encoding gene (FVEG_13172) was found to be highly induced by specific inhibitory Streptomyces strains and its deletion increased visible response to those strains. This study demonstrates that F. verticillioides does not have an all or nothing response to bacteria it encounters but rather a measured response that is strain specific and proportional to the strength of inhibition.

An N-acetylglucosamine transporter required for arbuscular mycorrhizal symbioses in rice and maize.

Most terrestrial plants, including crops, engage in beneficial interactions with arbuscular mycorrhizal fungi. Vital to the association is mutual recognition involving the release of diffusible signals into the rhizosphere. Previously, we identified the maize no perception 1 (nope1) mutant to be defective in early signalling. Here, we report cloning of ZmNope1 on the basis of synteny with rice. NOPE1 encodes a functional homologue of the Candida albicans N-acetylglucosamine (GlcNAc) transporter NGT1, and represents the first plasma membrane GlcNAc transporter identified from plants. In C. albicans, exposure to GlcNAc activates cell signalling and virulence. Similarly, in Rhizophagus irregularis treatment with rice wild-type but not nope1 root exudates induced transcriptome changes associated with signalling function, suggesting a requirement of NOPE1 function for presymbiotic fungal reprogramming.

Rice perception of symbiotic arbuscular mycorrhizal fungi requires the karrikin receptor complex.

In terrestrial ecosystems, plants take up phosphate predominantly via association with arbuscular mycorrhizal fungi (AMF). We identified loss of responsiveness to AMF in the rice (Oryza sativa) mutant hebiba, reflected by the absence of physical contact and of characteristic transcriptional responses to fungal signals. Among the 26 genes deleted in hebiba, DWARF 14 LIKE is, the one responsible for loss of symbiosis . It encodes an alpha/beta-fold hydrolase, that is a component of an intracellular receptor complex involved in the detection of the smoke compound karrikin. Our finding reveals an unexpected plant recognition strategy for AMF and a previously unknown signaling link between symbiosis and plant development.

Carbohydrate utilization and metabolism is highly differentiated in Agaricus bisporus.

BACKGROUND: Agaricus bisporus is commercially grown on compost, in which the available carbon sources consist mainly of plant-derived polysaccharides that are built out of various different constituent monosaccharides. The major constituent monosaccharides of these polysaccharides are glucose, xylose, and arabinose, while smaller amounts of galactose, glucuronic acid, rhamnose and mannose are also present. RESULTS: In this study, genes encoding putative enzymes from carbon metabolism were identified and their expression was studied in different growth stages of A. bisporus. We correlated the expression of genes encoding plant and fungal polysaccharide modifying enzymes identified in the A. bisporus genome to the soluble carbohydrates and the composition of mycelium grown compost, casing layer and fruiting bodies. CONCLUSIONS: The compost grown vegetative mycelium of A. bisporus consumes a wide variety of monosaccharides. However, in fruiting bodies only hexose catabolism occurs, and no accumulation of other sugars was observed. This suggests that only hexoses or their conversion products are transported from the vegetative mycelium to the fruiting body, while the other sugars likely provide energy for growth and maintenance of the vegetative mycelium. Clear correlations were found between expression of the genes and composition of carbohydrates. Genes encoding plant cell wall polysaccharide degrading enzymes were mainly expressed in compost-grown mycelium, and largely absent in fruiting bodies. In contrast, genes encoding fungal cell wall polysaccharide modifying enzymes were expressed in both fruiting bodies and vegetative mycelium, but different gene sets were expressed in these samples.

Polyphony in the rhizosphere: presymbiotic communication in arbuscular mycorrhizal symbiosis.

The Arbuscular Mycorrhizal (AM) symbiosis is a ubiquitous relationship established in terrestrial ecosystems between the roots of most plants and fungi of the Glomeromycota. AM fungi occur amongst many other inhabitants of the soil, and successful development of AM symbioses relies on a pre-symbiotic signal exchange that allows mutual recognition and reprogramming for the anticipated physical interaction. The nature of some of the signals has been discovered in recent years, providing a first insight into the type of chemical language spoken between the two symbiotic partners. Importantly, these discoveries suggest that the dialogue is complex and that additional factors and corresponding receptors remain to be unveiled. Here, we explore the latest advances in this pre-symbiotic plant-fungal signal exchange and present the resulting current understanding of rhizosphere communication in AM symbioses.

Assessment of autophagosome formation by transmission electron microscopy.

Autophagy is a complex degradative process by which cytosolic material, including organelles, is randomly sequestered within double-membrane bound vesicles termed autophagosomes and targeted for degradation. Initially described as a nutrient stress adaptation response, the process of autophagy is now recognized as a central mechanism involved in many developmental processes. In this chapter, we provide guidelines to assess the initial steps of autophagy by monitoring autophagic body vacuolar accumulation. We employed a standard electron microscopy approach to observe the vacuoles of nutrient stressed fungal cells.

The snf1 gene of Ustilago maydis acts as a dual regulator of cell wall degrading enzymes.

Many fungal plant pathogens are known to produce extracellular enzymes that degrade cell wall elements required for host penetration and infection. Due to gene redundancy, single gene deletions generally do not address the importance of these enzymes in pathogenicity. Cell wall degrading enzymes (CWDEs) in fungi are often subject to carbon catabolite repression at the transcriptional level such that, when glucose is available, CWDE-encoding genes, along with many other genes, are repressed. In Saccharomyces cerevisiae, one of the main players controlling this process is SNF1, which encodes a protein kinase. In this yeast, Snf1p is required to release glucose repression when this sugar is depleted from the growth medium. We have employed a reverse genetic approach to explore the role of the SNF1 ortholog as a potential regulator of CWDE gene expression in Ustilago maydis. We identified U. maydis snf1 and deleted it from the fungal genome. Consistent with our hypothesis, the relative expression of an endoglucanase and a pectinase was higher in the wild type than in the Δsnf1 mutant strain when glucose was depleted from the growth medium. However, when cells were grown in derepressive conditions, the relative expression of two xylanase genes was unexpectedly higher in the Δsnf1 strain than in the wild type, indicating that, in this case, snf1 negatively regulated the expression of these genes. Additionally, we found that, contrary to several other fungal species, U. maydis Snf1 was not required for utilization of alternative carbon sources. Also, unlike in ascomycete plant pathogens, deletion of snf1 did not profoundly affect virulence in U. maydis.

The autophagy genes ATG8 and ATG1 affect morphogenesis and pathogenicity in Ustilago maydis.

Autophagy is a complex degradative process in which cytosolic material, including organelles, is randomly sequestered within double-membrane vesicles termed autophagosomes. In Saccharomyces cerevisiae, the autophagy genes ATG1 and ATG8 are crucial for autophagy induction and autophagosome assembly, respectively, and their deletion has an impact on the autophagic potential of the corresponding mutant strains. We were interested in the role of autophagy in the development and virulence of U. maydis. Using a reverse genetic approach, we showed that the U. maydis ATG8 orthologue, atg8, is associated with autophagy-dependent processes. Deletion of atg8 abolished autophagosome accumulation in the vacuoles of carbon-starved cells and drastically reduced the survival of U. maydisDeltaatg8 mutant strains during these conditions. In addition, atg8 deletion had an impact on the budding process during saprobic haploid growth. The infection of maize with compatible Deltaatg8 strains resulted in fewer galled plants, and fungal gall colonization was strongly reduced, as reflected by the very low hyphal density in these tissues. Deltaatg8 infections resulted in the formation of very few teliospores. To corroborate the role of autophagy in U. maydis development, we also deleted the ATG1 orthologue, atg1. Deletion of atg1 yielded phenotypes similar to the Deltaatg8 strains during saprobic growth, but of lower magnitude. The Deltaatg1 strains were only slightly less pathogenic than the wild-type and teliospore production was not affected. Surprisingly, atg1 deletion in the Deltaatg8 background exacerbated those phenotypes already observed in the Deltaatg8 and Deltaatg1 single-mutant strains, strongly suggesting an additive phenotype. In particular, the double mutant was completely suppressed for plant gall induction.

DelsGate: a robust and rapid method for gene deletion.

Gene deletion is one of the most powerful tools to study gene function. In the genomics era there is great demand for fast, simple high-throughput methods for gene deletion to study the roles of the large numbers of genes that are being identified. Here we present an approach that speeds up the process of generation of deletion mutants by greatly simplifying the production of gene deletion constructs. With this purpose we have developed a method, which we named DelsGate (Deletion via Gateway), that combines PCR and Gateway cloning technology together with the use of the I-SceI homing endonuclease to generate precise deletion constructs in a very simple, universal and robust manner in just 2 days. DelsGate consists of standard PCR of only the 5' and 3' 1 kb gene flanks directly followed by in vitro Gateway cloning and final generation of the circular deletion construct by in vivo recombination in Escherichia coli. For use in DelsGate we have modified a Gateway cloning vector to include selectable markers for the transformation of Ascomycetes and the Basidiomycete fungus Ustilago maydis. The PCR and transformation steps of DelsGate should be well suited for high-throughput approaches to gene deletion construction in fungal species. We describe here the entire process, from the generation of the deletion construct with DelsGate to the analysis of the fungal transformants to test for gene replacement, with the Basidiomycete fungus Ustilago maydis. Application of DelsGate to other fungal species is also underway. Additionally, we describe how this basic approach can be adapted to other genetic manipulations with minor changes. We specifically describe its application to create unmarked deletions in Ralstonia solanacearum, a Gram-negative phytopathogenic bacterium.

Dimorphism in fungal plant pathogens.

Fungi are mostly sessile organisms, and thus have evolved ways to cope with environmental changes. Many fungi produce 'dormant' structures, which allow them to survive periods of unfavorable conditions. Another ingenious active approach to a changing environment has been adopted by the 'dimorphic fungi', which simply shift their thallic organization as a way to adapt and thrive in the new conditions. Dimorphism is extensively exploited by both plant and animal pathogenic fungi, where the encounter with the host prompts a shift in the mode of growth. In this review, we focus on the phenomenon of dimorphism among plant pathogenic fungi through discussion of several relatively well-studied exemplar species.

Sending mixed signals: redundancy vs. uniqueness of signaling components in the plant pathogen, Ustilago maydis.

The ability to respond to a changing environment separates successful organisms from their competitors. Thus, signal transduction is a crucial aspect of an organism's growth, development, differentiation, and reproduction. Nowhere is this more evident than in the co-evolution of obligate pathogens with their host organisms. The genome sequence of Ustilago maydis, the pathogen of maize, has provided a powerful tool in the assessment and characterization of signaling pathways for this organism. Inspection of the sequence reveals that while U. maydis has a streamlined gene content, it appears to contain a full repertoire of the standard signaling cascades present in other fungi. A full range of paralogues are present to provide redundancy of function on the one hand while, on the other, distinct strategies for survival. This review explores signaling based on the conserved mitogen-activated protein (MAP) kinase and cAMP-dependent protein kinase A (PKA) pathways as well as ancillary functions, with emphasis on the unique aspects of the U. maydis approach to utilizing this architecture.

DelsGate, a robust and rapid gene deletion construction method.

With the increasing availability of fungal genome sequences there is great demand for fast, simple high-throughput methods to generate constructs for gene deletion. Here we describe a method that combines PCR and Gateway cloning technology together with use of the I-SceI homing endonuclease to generate precise deletion constructs in a very simple, universal and robust manner in just 2 days. These constructs are then used to produce deletion mutants in the organism of interest following applicable methods for that species. In establishing this protocol we determined empirically that 1 kb was a suitable flank length to facilitate homologous recombination in our species of interest, Ustilago maydis. The method, which we have named DelsGate (Deletions via Gateway), consists of standard PCR of only the 5' and 3' 1 kb gene flanks directly followed by in vitro Gateway cloning and final generation of the circular deletion construct by in vivo recombination in Escherichia coli. For use in DelsGate we have modified a Gateway cloning vector to include selectable markers for transformation of Ascomycetes and the Basidiomycete fungus U. maydis which causes corn smut disease. We have tested the reproducibility of the DelsGate approach by generating deletion constructs for 12 U. maydis genes. Although not tested here, the PCR and transformation steps of DelsGate should be well suited for high-throughput approaches to gene deletion construction in fungal species. DelsGate has the potential to be universal for all organisms with efficient transformation and homologous recombination systems.

Sex in broad daylight: turning a new leaf-fungal style.

Cryptococcus spp. are potentially lethal fungal pathogens of humans and animals that are not easily communicable. In this issue of Cell Host & Microbe, Xue et al. describe sexual reproduction of Cryptococcus in association with plants and identify plant-derived mating stimulatory compounds. These results may have wide significance in explaining environmental sources of effective inoculum in human disease foci.