Research in Plant Pathology

and Fungal Genetics

Where We Are

Welcome to the Vaillancourt laboratory informational web page! Our home is at the University of Kentucky in Lexington. Lexington is a beautiful place to live, in the heart of the bluegrass region famous for its thoroughbred horse farms and horse-racing. The Department of Plant Pathology is located in the new Plant Science Building, on the south end of the UK campus.

Introduction to our Research

Our goal is to understand how a pathogenic fungus and its host plant communicate with each other during a disease interaction. Disease symptoms (lesions, rotting) typically result from activities of both the host plant and the fungal pathogen. These activities are regulated by the communication (molecular signaling) that occurs between the two. There are numerous examples of "pathogenic dimorphism" in which the host-pathogen association alternates between relatively non-destructive (latent, endophytic, biotrophic) and destructive (necrotrophic). In our lab, we are focused on elucidating the molecular basis for pathogenic dimorphism, as a way to better understand the fundamental basis of communication between host and pathogen that leads to disease.

Our primary research focus is on the pathogenic fungual genus Colletotrichum, which cause anthracnose diseases and fruit rots on various host plants. Colletotrichum fungi are typically hemibiotrophic, which means that they are initially biotrophic (or latent) and later switch to a destructive necrotrophic mode accompanied by extensive rotting of the host tissues. In our lab we have worked mostly with C. graminicola, which causes anthracnose disease of corn. Symptoms of corn anthracnose include lesions on the leaves and rotting of the stalk tissues. We have specifically been trying to clone and characterize fungal genes that are involved in the ability to sense and respond to the host environment, and/or that are required for destructive (necrotrophic) growth in planta. We have recently begun to study another Colletotrichum pathosystem on Arabidopsis in collaboration with Dr. Pradeep Kachroo in our department. We hope to be able to understand more about the plant molecular signaling related to pathogenic dimorphism by taking advantage of the model plant Arabidopsis.

We are also studying the asexual fungus Sphaeropsis sapinea which causes pine tip blight disease on two-needle pines. S. sapinea has a latent phase durng which it colonizes the host tissues, but does not trigger symptoms. We are interested in understanding the relationship between stress in the host tree and switching of the fungus from latent to pathogenic (rotting) growth and symptom development.


Life Cycle of Colletotrichum graminicola

Colletotrichum graminicola is a filamentous fungus in the ascomycete group, the same class as the better known pink bread mold fungus, Neurospora crassa. C. graminicola is an ideal organism for our genetic studies because we can cross different strains to study segregation of genes, and we can also manipulate genes directly using standard molecular biological techniques.

Asexual Reproduction

The life cycle of C. graminicola is fairly typical for its group. Vegetative spores (resulting through the process of mitosis) are produced in large numbers on plants or on artificial media. These are banana-shaped (or falcate) and contain a single nucleus. The spores develop on a cushion-shaped structure just below the plant's epidermis. Each spore is "blown out" from a specialized peg-like structure called a conidiophore. The spores eventually erupt through the surface of the plant, and the resulting fruiting body is called an acervulus. Scattered among the spores in the acervulus are numerous dark hair-like structures called setae. The spores are contained in a mucilagenous, water-soluble matrix. Molecular regulation of the process of conidiation was the particular interest of the late Dr. Robert Hanau. Dr. G-C (Eric) Fang recently published research that he conducted in collaboration with Dr. Hanau and with our laboratory, demonstrating a role of reactive oxygen species and for a manganese-type superoxide dismutase in the process of conidiation.

A single spore, when carried to a new plant by splashing rain or placed on nutrient medium, will germinate and produce a thread-like structure called a hypha. The very first hypha is called a germ tube. The hypha grows apically, and branches frequently. The eventual result is a mass of hyphae called a mycelium.

When the hyphae grow submerged in host tissues, or under the surface of an agar medium, they produce numerous vegetative spores of a second type. These are oval in shape, and are budded off the hyphae rather like yeast cells. These spores can germinate and develop into a mycelium in the same way as the banana-shaped spores, which are produced only on the surface.

Spore Germination

Germination of the falcate spores occurs after a period of dormancy. We have been studying the environmental cues that contribute to this event, and we find that surface hydrophobicity and surface rigidity can both induce spore germination, in the absence of nutrients. If a source of carbon is present, the spore will germinate regardless of surface characteristics. Oval spores do not respond to environmental cues in the same way. We are very interested in the signaling pathways that are involved in the ability of the spore to sense its surroundings, and in the germination response to surface cues.

Sexual Reproduction

Given certain environmental conditions that we do not yet fully understand, individual C. graminicola strains or pairs of strains produce sexual structures called perithecia. These are flask-shaped organs which contain the sexual spores (arising through the process of meiosis). The sexual phase of the life cycle of C. graminicola has its own name, Glomerella graminicola. The sexual spores of G. graminicola, like those of other ascomycetes, are called ascospores and are contained in sack-like structures called asci. Each ascus contains eight spores. All eight spores (called a tetrad) are the result of a single meiosis, followed by a mitotic division. Tetrads can be isolated from G. graminicola, allowing individual meioses to be studied directly. Alternatively, random spores can be collected for genetic analyses as they are exuded in a mucilagenous matrix from the necks of the perithecia. Ascospores germinate and produce a mycelium in the same way as either type of vegetative spore.

We have been studying the genetics of sexual reproduction in G. graminicola, and we know that the regulation of this process is very complex. Certainly, more than a single locus regulates intercompatibility among strains. G. graminicola appears to be fundamentally homothallic, but many self-sterile strains exist, and most of these appear to be cross-fertile with most other self-sterile or self-fertile strains. Crosses in Glomerella proceed in a similar fashion to true heterothallic matings among other fungi, with one of the mates acting as the nuclear acceptor (female) and the other as the nuclear donor (male). The female strain produces the perithecial wall, and all of the progeny within the perithecium have the same mitochondrial DNA type as the maternal parent. Self-fertile strains are extremely unstable, and produce self-sterile progeny or sectors frequently. We have recently cloned a putative mating-type gene from G. graminicola, which is similar to MAT-2 idiomorph loci that have been described from other fungi. Our next goal will be to determine if this gene functions in fertility or sexuality in Glomerella.

Infection of Corn by Colletotrichum graminicola

Colletotrichum graminicola, like some other plant pathogenic fungi, produces a specialized cell called an appressorium which it uses to force its way into the cells of a corn plant. When a spore germinates, the tip of the germ tube adheres tightly to the surface it is resting on. If the surface is firm, like a plant cell wall, the hypha responds by stopping apical growth. The tip of the hypha begins to swell. Soon, a wall is formed inside the germ tube that isolates the swollen tip. This swollen tip will become the appressorium. Eventually, the swelling stops and the fungus begins to deposit a dark pigment called melanin in the walls of the appressorium. Melanin is important because it allows the fungus to build up extremely high turgor pressures within the appressorial cell. It acts something like a one-way valve, which allows water in but doesn't allow solutes (particularly a sugar called glycerol) out. Pressures within appressorial cells have been estimated to exceed those found in a standard automobile tire, and may be the highest found anywhere in a living cell. Only one spot in the appressorial wall doesn't have any melanin, and this is the area that is in contact with the corn cell wall. This area is called the germination pore. Eventually, the pressures build up enough so that the fungus can force a hypha (called a penetration peg) through the germination pore and right through the corn cell wall. C. graminicola also uses a mixture of enzymes to soften the plant cell wall. Once inside, the penetration peg swells and grows through the plant tissues, eventually resulting in the death of the plant cells.

REMI mutagenesis

We used random mutagenesis in order to identify genes that are important for colonization of corn pith tissues. We used a technique called restriction-fragment mediated insertional mutagenesis, or REMI mutagenesis to produce nearly 2000 (and counting) randomly mutated strains. All of these strains are stored permanantly on silica in our freezer. We have screened nearly 1600 of these for their pathogencity to corn leaves and wounded corn pith tissue. We have identified nine mutants that are either completely nonpathogenic to stalks and leaves of corn, or that are significantly reduced in pathogenicity. We are currently in the process of characterizing these mutants.

One of the nine pathogenicity mutants has recently become the focus of our research. This mutant causes no symptoms on either leaves or stalks of corn. The strain is mutated in a gene that is predicted to encode one subunit of the microsomal signal peptidase. The signal peptidase is responsible for cleavage of signal peptides from proteins as they are transported across the endoplasmic reticulum membrane. With our collaborator Dr. Charles Mims of the university of Georgia, we completed an ultrastructural comparison of infection processes of the mutant and wild type strains. We found that the mutant appears to be specifically deficient in the ability to transform from biotrophic to necrotrophic growth. Our current hypothesis is that the mutant is unable to secrete sufficient quantities of one or more proteins that are required to initiate or to sustain necrotrophic growth. Our current focus is on testing the hypothesis that CPR1 does encode a component of the signal peptidase, and on understanding the basis for the pathogenicity defect in this mutant.