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Given a shared toolbox of pathogenicity-related genes among a set of species, why is one species a biotroph and specialist while another is a necrotroph and generalist? Is it the result of selection on primary sequence, or on proteins, or alternatively, differences in the timing and magnitude of gene expression? The Sclerotiniaceae (Ascomycota, Leotiomycetes, Helotiales) is a relatively recently evolved family of fungi whose members include host generalists and host specialists, and the spectrum of trophic types. Based on a phylogeny inferred from three, presumably evolutionarily conserved housekeeping genes, the common ancestor of the Sclerotiniaceae was necrotrophic, with at least two shifts from necrotrophy to biotrophy. Phylogenies inferred from eight pathogenicity-related genes, involved in cell wall degradation and the oxalic acid pathway, were incongruent with the presumably neutral phylogeny. Site-specific likelihood analyses, which estimate the rate of nonsynonymous to synonymous substitutions (dN/dS), showed evidence for purifying selection acting on all pathogenicity-related genes, and positive selection on sites within five of eight genes. Rate-specific likelihood analyses showed no differences in dN/dS rates between necrotrophs and biotrophs, and between host generalists and host specialists, indicating that selection acting on the genes does not drive divergence toward changes in trophic type or host association. In vitro screens for oxalic acid production demonstrated that all necrotrophic generalists produce oxalic acid by 72 hours, while production was either absent or delayed among biotrophs and host specialists. This pattern was also observed during the course of Arabidopsis thaliana infection, in which large spikes of expression were seen in the oxalic acid pathway-related gene, oah, within eight hours of inoculation among necrotrophic generalists only. Results suggest that necrotrophic generalists can be distinguished from biotrophs and host specialists in the Sclerotiniaceae by the ability to produce abundant amounts of oxalic acid early in infection and to cause large proliferating lesions on A. thaliana.

Given a shared toolbox of pathogenicity-related genes among a set of species, why is one species a biotroph and specialist while another is a necrotroph and generalist? Is it the result of selection on primary sequence, or on proteins, or alternatively, differences in the timing and magnitude of gene expression? The Sclerotiniaceae (Ascomycota, Leotiomycetes, Helotiales) is a relatively recently evolved family of fungi whose members include host generalists and host specialists, and the spectrum of trophic types. Based on a phylogeny inferred from three, presumably evolutionarily conserved housekeeping genes, the common ancestor of the Sclerotiniaceae was necrotrophic, with at least two shifts from necrotrophy to biotrophy. Phylogenies inferred from eight pathogenicity-related genes, involved in cell wall degradation and the oxalic acid pathway, were incongruent with the presumably neutral phylogeny. Site-specific likelihood analyses, which estimate the rate of nonsynonymous to synonymous substitutions (dN/dS), showed evidence for purifying selection acting on all pathogenicity-related genes, and positive selection on sites within five of eight genes. Rate-specific likelihood analyses showed no differences in dN/dS rates between necrotrophs and biotrophs, and between host generalists and host specialists, indicating that selection acting on the genes does not drive divergence toward changes in trophic type or host association. In vitro screens for oxalic acid production demonstrated that all necrotrophic generalists produce oxalic acid by 72 hours, while production was either absent or delayed among biotrophs and host specialists. This pattern was also observed during the course of Arabidopsis thaliana infection, in which large spikes of expression were seen in the oxalic acid pathway-related gene, oah, within eight hours of inoculation among necrotrophic generalists only. Results suggest that necrotrophic generalists can be distinguished from biotrophs and host specialists in the Sclerotiniaceae by the ability to produce abundant amounts of oxalic acid early in infection and to cause large proliferating lesions on A. thaliana.

Doctor of Philosophy / Department of Plant Pathology / Barbara S. Valent / Rice blast caused by the ascomycetous fungus Magnaporthe oryzae remains a threat to global sustainable agriculture and food security. This pathogen infects staple cereal crops such as rice, wheat, barley and millets, as well as turf grasses, in a distinct way among fungal plant pathogens, which we described in the first chapter. In addition to economical importance, rice blast is a model pathosystem for difficult-to-study biotrophic fungi and fungal-plant interactions. We are studying proteins that fungi secrete inside living cells to block plant defenses and control host cell processes; these proteins are called effectors. To date mechanisms for secretion and delivery of effectors inside host cells during disease establishment remain unknown. This step is critical to ensure the successful infection. So far, the only commonality found among all unique small-secreted blast effector proteins is their accumulation in a novel in planta structure called the biotrophic-interfacial complex (BIC). Identifying effectors and understanding how they function inside rice cells are important for attaining durable disease control. In the second chapter, we presented one approach to address this challenge. We characterized four candidate effector genes that were highly expressed specifically during the rice cell invasion. Using transgenic fungi that secrete fluorescently-labeled versions of each protein allowed me to follow them during invasion in vivo by live cell imaging. These candidates show distinct secretion patterns suggesting a spatially-segregated secretion mechanism for effectors. Results revealed a BIC-located strong candidate cytoplasmic blast effector, two putative cell-to-cell movement proteins and a putative extrainvasive hyphal membrane (EIHM)-matrix protein, which has become a valuable tool for assessing successful infection sites. In the third chapter, we test if normal secretion components of filamentous fungi are involved in accumulation of effectors into BICs. We report localization studies with M. oryzae orthologs of conserved secretion machinery components to investigate secretion mechanisms for effectors showing preferential BIC accumulation and for non-BIC proteins such as BAS4. Especially bright fluorescence adjacent to BICs from Mlc1p (Myosin Light Chain, a Spitzenkörper marker), from Snc1p (a secretory vesicle marker), and from Yup1p (a putative t-SNARE endosomal protein) suggest secretion actively occurs in the BIC-associated cells. Localization of Spa2p (a polarisome marker), as a distinct spot at the tips of the bulbous invasive hyphae (IH) in planta, suggests the existence of two secretion complexes after the fungus switches growth from the polarized filamentous primary hyphae to bulbous IH. In the final chapter on future perspectives, we present some strategies towards the molecular understanding of the M. oryzae secretion mechanism during biotrophic invasion, which will lead to novel strategies for disease control.

Endophytic fungi inhabit the living tissue of a host plant for at least a portion of their life cycle. While some researchers have shown that various endophytic fungi participate in litter decomposition, we do not know whether such fungi are actually saprotrophic, meaning that they can obtain energy from litter. Therefore, I determined if endophytic fungi are saprotrophs using leaf litter as the energy source. All 49 tested isolates were found to be saprotrophic. To compare the saprotrophic capacities of fungi from different habitats, which produce different types of litter, a universal litter proxy needs to be used. I hypothesized that pure cellulose would be an adequate proxy for litter for in vitro studies because of its abundance in litter. This was tested in the first study. Saprotrophic capacity on pure cellulose was not highly correlated with that on leaf litter. I conclude, therefore, that cellulose may not be a good proxy for leaf litter. Some endophytic fungi are biotrophs, presumably acquiring energy from photosynthate produced by the host plant. This suggests that the level of exposure to sunlight by the plant should influence the competitive ability of such fungi. If saprotrophic endophytic fungi do exist, they ought to be less competitive against biotrophic endophytic fungi in leaves receiving full sunlight than in shaded leaves. I, therefore, hypothesized that the frequency of saprotrophy will be influenced by the level of sun exposure of the leaf from which the fungi were isolated. This was tested in the second study. Moreover, because closely related organisms ought to be more similar to each other than more distantly related organisms, I also hypothesized that saprotrophic capacity has a strong phylogenetic component, which was also tested in the second study. Unexpectedly, isolate identity within genus accounted for far more variability in saprotrophic capacity than genus identity, and sun exposure did not have a significant effect on saprotrophy. These results suggest that saprotrophic capacity may not be highly consequential in the ecology of these organisms.

Doctor of Philosophy / Department of Plant Pathology / Barbara S. Valent / Rice blast is a major fungal disease on rice, caused by the hemibiotrophic filamentous ascomycete fungus, Magnaporthe oryzae. This disease accounts for 157 million tons of grain loss annually. The fungus produces a specialized cell called appressorium to penetrate the host surface barrier and enter inside. It produces intracellular Invasive Hyphae (IH) that grow form cell to cell to colonize the host. The mechanisms of appressorium formation and host penetration have been studied in detail but the host colonization strategies remain largely unknown. We applied live-cell imaging to characterize spatial and temporal development of IH and plant responses inside successively-invaded rice cells. Early loading experiments with the endocytotic tracker, FM4-64, showed dynamic plant membranes around IH. These hyphae showed remarkable plasticity and recruited plant cell components. IH exhibited pseudohyphal growth and were sealed in plant membrane, termed the Extra-Invasive Hyphal Membrane (EIHM). The fungus spent up to 12 hours in the first cell, often tightly packing it with IH. IH that moved into neighboring cells were biotrophic, although they were initially thinner and grew more rapidly. IH in neighboring cells were wrapped in EIHM with distinct membrane caps at the hyphal tips. Time-lapse imaging showed IH scanning plant cell walls before crossing them, and transmission electron microscopy showed crossing occurring at pit fields. This and additional evidence strongly suggest that IH co-opt plasmodesmata for cell-to-cell movement. Our studies have revealed insights into a novel hemibiotrophic strategy employed by the blast fungus. Few genes have been previously characterized that impact the biotrophic IH. To understand the molecular basis of the biotrophic infection strategy we employed Laser Microdissection (LM) technology to isolate and purify the IH at this early growth stage. We compared the gene expression of these samples with axenically-grown mycelium using M. oryzae whole genome microarrays. We identified several hundreds of infection specific genes. We have shown that LM technology can be used to isolate homogenous cells from the infected rice tissues to study the underlying molecular mechanisms of signaling during disease formation. These studies will be very critical to understand the host-pathogen interactions to eventually develop durable management strategies.