The growth of wild-type and genetically-marked strains of Tilletia caries and T. controversa in susceptible and resistant wheat cultivars

The objective of this study was to provide basic information on the interaction of the bunt fungi Tilletia caries and T. controversa with their host, Triticum aestivum. Studies on the ability of haploid strains to colonize wheat heads provided the basis for experiments in which haploid strains were crossed in planta. Wild-type and genetically-marked haploid strains of T. caries and T. controversa injected into boot-stage heads of susceptible and resistant wheat cultivars were recovered from the mature heads 4 to 6 weeks after introduction. Viable teliospores were produced following successful matings of sexually compatible haploid cultures of T.caries and T.controversa injected into boot-stage heads which were grown both in intact plants and in an organ culture system for wheat heads. The organ culture system adopted here was determined to simulate natural infection under controlled environmental conditions. It served as a basis for the production of hybrid teliospores formed between strains of T. caries and T. controversa. The kinetics of teliospore germination of T. caries, T. controversa and hybrids formed by the cross of T. caries, and a genetically-marked strain of T. controversa, were determined. The time required for germination of the hybrid teliospores at 4C was intermediate to that of the parents, suggesting that this trait may be controlled by a single gene which exhibits incomplete dominance. Teliospores of the Fl generation failed to germinate at 18C suggesting that this trait is controlled by a dominant gene present in the T. controversa parental strain. The genetic marker was recovered in the Fl generation, indicating that drug-resistance markers may be used in future crosses

Optimized primers and other critical conditions for efficient fusion PCR to generate knockout vectors in filamentous fungi.

Methods to streamline functional studies of large numbers of genes are essential to fully utilize the significant genomic resources now available for fungi. Fusion PCR is often used to join pieces of DNA together, particularly in the construction of DNA fragments for gene replacement in fungi. Here we present high-efficiency primers which reliably direct fusion and amplification to generate constructs for gene knockouts. Addendum: The authors communicated this omission post publication: We inadvertently left out the 3HS sequence with which we had such great success. The 5HS sequence, as seen in Figure 2D, is 5\u27-AGTCGACGACAACTACCATCGATCTGACG. The 3HS sequence which was missing from the original paper is 5\u27-ACACTGGTGACGGCTAACCAGAACTGTCA

The Fast-Evolving phy-2 Gene Modulates Sexual Development in Response to Light in the Model Fungus Neurospora crassa

Rapid responses to changes in incident light are critical to the guidance of behavior and development in most species. Phytochrome light receptors in particular play key roles in bacterial physiology and plant development, but their functions and regulation are less well understood in fungi. Nevertheless, genome-wide expression measurements provide key information that can guide experiments that reveal how genes respond to environmental signals and clarify their role in development. We performed functional genomic and phenotypic analyses of the two phytochromes in Neurospora crassa, a fungal model adapted to a postfire environment that experiences dramatically variable light conditions. Expression of phy-1 and phy-2 was low in early sexual development and in the case of phy-2 increased in late sexual development. Under light stimulation, strains with the phytochromes deleted exhibited increased expression of sexual development-related genes. Moreover, under red light, the phy-2 knockout strain commenced sexual development early. In the evolution of phytochromes within ascomycetes, at least two duplications have occurred, and the faster-evolving phy-2 gene has frequently been lost. Additionally, the three key cysteine sites that are critical for bacterial and plant phytochrome function are not conserved within fungal phy-2 homologs. Through the action of phytochromes, transitions between asexual and sexual reproduction are modulated by light level and light quality, presumably as an adaptation for fast asexual growth and initiation of sexual reproduction of N. crassa in exposed postfire ecosystems

The power of discussion : Support for women at the fungal Gordon Research Conference

We would like to thank Abigail LaBella for sharing the pictures taking during the session and Felicia Wu for her suggestions. We are grateful to all the colleagues that helped leading the discussion groups and all the participants of the session.Peer reviewedPostprin

Global gene expression and knockout analysis reveals genes associated with fungal fruiting body development in Neurospora crassa

ungi can serve as highly tractable models for understanding genetic basis of sexual development in multicellular organisms. Applying a reverse-genetic approach to advance such a model, we used random and multitargeted primers to assay gene expression across perithecial development in Neurospora crassa. We found that functionally unclassified proteins accounted for most upregulated genes, whereas downregulated genes were enriched for diverse functions. Moreover, genes associated with developmental traits exhibited stage-specific peaks of expression. Expression increased significantly across sexual development for mating type gene mat a-1 and for mat A-1 specific pheromone precursor ccg-4. In addition, expression of a gene encoding a protein similar to zinc finger, stc1, was highly upregulated early in perithecial development, and a strain with a knockout of this gene exhibited arrest at the same developmental stage. A similar expression pattern was observed for genes in RNA silencing and signaling pathways, and strains with knockouts of these genes were also arrested at stages of perithecial development that paralleled their peak in expression. The observed stage specificity allowed us to correlate expression upregulation and developmental progression and to identify regulators of sexual development. Bayesian networks inferred from our expression data revealed previously known and new putative interactions between RNA silencing genes and pathways. Overall, our analysis provides a fine-scale transcriptomic landscape and novel inferences regarding the control of the multistage development process of sexual crossing and fruiting body development in N. crassa

The L-Type Calcium Ion Channel Cch1 Affects Ascospore Discharge and Mycelial Growth in the Filamentous Fungus Gibberella zeae (Anamorph Fusarium graminearum)▿ †

Cch1, a putative voltage-gated calcium ion channel, was investigated for its role in ascus development in Gibberella zeae. Gene replacement mutants of CCH1 were generated and found to have asci which did not forcibly discharge spores, although morphologically ascus and ascospore development in the majority of asci appeared normal. Additionally, mycelial growth was significantly slower, and sexual development was slightly delayed in the mutant; mutant mycelia showed a distinctive fluffy morphology, and no cirrhi were produced. Wheat infected with Δcch1 mutants developed symptoms comparable to wheat infected with the wild type; however, the mutants showed a reduced ability to protect the infected stalk from colonization by saprobic fungi. Transcriptional analysis of gene expression in mutants using the Affymetrix Fusarium microarray showed 2,449 genes with significant, twofold or greater, changes in transcript abundance across a developmental series. This work extends the role of CCH1 to forcible spore discharge in G. zeae and suggests that this channel has subtle effects on growth and development

Deoxynivalenol Biosynthesis-Related Gene Expression During Wheat Kernel Colonization by \u3ci\u3eFusarium graminearum\u3c/i\u3e

Deoxynivalenol (DON) is a potent mycotoxin and virulence factor produced by Fusarium graminearum. We examined the expression of the core DON biosynthetic gene Tri5 during wheat head infection of susceptible and resistant cultivars and susceptible cultivars treated with strobilurin fungicides (e.g. azoxystrobin). DON was quantified to correlate expression with toxin accumulation. The highest Tri5 expression relative to housekeeping genes occurred at the infection front. As infection progresses, earliest-infected kernels showed diminished relative Tri5 expression but Tri5 expression never ceased during the 21 days observed. Azoxystrobin treatment showed no significant effect on either relative Tri5 expression or DON quantity. The resistant cultivar \u27Alsen\u27 showed minimal spread of the fungus, with no fungus detected by day 21. DON was not detected in significant quantities in Alsen in the later stages sampled. In Wheaten, DON levels were negligible at 8 days post-inoculation (dpi), with detectable DON at later-sampled time points. Tri5 was detected even in fully senesced kernels 21 dpi. Our data demonstrate the presence of Tri5 transcripts in a susceptible cultivar over a much longer time period than has been previously documented. This suggests the ability of the fungus to rapidly resume toxin biosynthesis in dried infected grain should conducive environmental conditions be present, and provides a possible mechanism for high DON levels in asymptomatic grain