University of Kentucky College of Agriculture

THE VAILLANCOURT LAB

Research in Plant Pathology

and Fungal Genetics

the labWelcome 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.


Our Research

In our laboratory, we are interested in the “rot” diseases of plants, particularly corn stalk rots, and anthracnose rots and leaf blights of various plants, including the model plant Arabidopsis. The fungi that cause rots are remarkably successful pathogens, and rots are among the most difficult and expensive diseases to manage using conventional approaches. Many are associated with plant senescence and physiological ripening, when host genetic resistance mechanisms appear to lose much of their efficacy. Although rots are often thought of as rather unsophisticated, especially when compared with the more elegant biotrophs, we know that rotting (i.e. dissolution of plant cell walls by fungal cell wall degrading enzymes) is actually a highly regulated process that does not occur at all times or in all tissues. We are using a molecular genetic approach to try to understand symptom development and host-pathogen interactions in rot diseases.

Corn Stalk Rot

Stalk rot is one of the most economically important diseases of corn worldwide. Industry estimates suggest that up to 6% of our potential corn yield is lost to stalk rot, mainly because of reduced kernel weights. Complete control is currently impossible, although damage can usually be reduced by use of hybrids with partial resistance to one or more of the causal fungi, and by minimizing plant stress. Our long-term goal is to develop a detailed molecular and cellular understanding of the interactions between corn and corn stalk rot fungi so that we can manage stalk rot disease more effectively. As research subjects, we’ve chosen the two most common and serious causes of fungal stalk rot of corn: Colletotrichum graminicola and Gibberella zeae (= Fusarium graminearum). Both are excellent model organisms for genetic research. The complete genomes of both species are available from the Fungal Genome Initiative at the Broad Institute.

C. graminicola causes anthracnose stalk rot (ASR), leaf blight (ALB), and seedling blight of corn. Resistance to C. graminicola is primarily quantitative, although a few sources of major gene resistance have been described. Resistance to ASR is not always correlated with resistance to ALB, suggesting that mechanisms of resistance may be tissue specific. G. zeae causes Gibberella stalk rot (GSR) and ear rot of corn. Resistance of corn to G. zeae is also quantitative. Resistance to GSR is not necessarily correlated with resistance to ASR. G. zeae produces mycotoxins in infected tissues, whereas C. graminicola is not known to produce toxins. G. zeae apparently enters corn ears via the silks, and both G. zeae and C. graminicola are believed to invade intact stalk tissues through the stalk rind, roots, and/or leaf sheaths.

Generally speaking, anything that stresses the plants can increase corn stalk rot incidence. Precisely how stress affects disease severity is not understood, however. For example, nitrogen levels are important, but have a somewhat unpredictable effect on stalk rot severities. This suggests a complex relationship not just related to plant stress but perhaps also to the ability of the fungus to sense and utilize nitrogen in planta. A dominant hypothesis holds that drawing of carbohydrate reserves from stalks during grain fill causes increased susceptibility to stalk rot. This may be due to an associated reduction in natural host defenses, and/or to fungal sensing of changing nutrient status, since fungi are known to be very responsive to carbon quality and quantity. Understanding the molecular relationship of the stalk rot fungi to the nutritional and defensive status of the host is a goal for our research.

Disease symptoms (including 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 also working to elucidate the molecular basis for pathogenic dimorphism between rotting and non-rotting phases in both C. graminicola and G. zeae, as a way to better understand the fundamental basis of communication between host and pathogen that leads to disease.

The Genus Colletotrichum

The pathogenic fungal genus Colletotrichum 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 and Dr. Aardra 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.

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.

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 primarily on the surface.

Spore Germination

Germination of the falcate spores occurs after a period of dormancy. We have studied 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 studied 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.


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.

Colonization of Corn Leaves and Stalks

Fungal pathogens of plants can be classified as biotrophs, which feed only on living plant tissues, or necrotrophs, which live primarily on dead or dying cells. A majority of Colletotrichum fungi, including C. graminicola, are intracellular hemibiotrophs, an interesting third category that is biotroph-like or necrotroph-like during successive phases of their disease cycles. During biotrophic development of C. graminicola, initial penetration of a host cell is followed by production of a swollen primary infection hypha. Within a few hours, branches emerge and begin to infect other host cells via narrow connections through intact host cell walls. Newly infected cells remain alive while the host plasma membrane expands and becomes invaginated around these primary hyphae. Approximately 24 hours after infection, the plasma membrane of an invaded cell loses its functional integrity, and the cell begins to die. Cell death is gradual and confined to cells that have been infected by hyphae, and there is no extensive dissolution of the host cell walls at this point. Primary infection hyphae continue to colonize new cells, expanding the infection front until the more destructive necrotrophic phase begins, for C. graminicola 24-48 hours after initial infection. This phase is characterized by production of large quantities of extracellular lytic enzymes and numerous narrow secondary hyphae that ramify through the host tissues both inter- and intracellularly. Host cells are killed rapidly, and host cell walls become extensively degraded and rotted. It is only at this stage that the macroscopic symptoms of anthracnose begin to appear. The developmental transition between biotrophic and necrotrophic growth is an interesting and compelling problem, and also of great practical importance to the pathology of these organisms, since it is only during necrotrophic development that significant damage occurs, and reproduction can only occur on dead host tissues during the necrotrophic phase. Evidence in the literature suggests a connection to host defenses and host nutritional status.

It has been suggested that C. graminicola behaves as a wilt fungus, colonizing and moving through the xylem, leading to dieback of the upper parts of the stem. We used C. graminicola strains expressing green fluorescent proteins (GFPs) to observe colonization of wounded corn stalks. We found no evidence that C. graminicola is a wilt, but instead we observed that it efficiently colonizes and moves through the fiber cells that surround the vascular bundles, and that also underlie the epidermal cells in the stalk rind. Our work suggests that fiber colonization is an important feature of the pathogenicity of this fungus. Movement through the mostly non-living fibers may allow the fungus to avoid host defenses, providing a base from which it can invade adjacent parenchyma 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 randomly mutated strains. All of these strains are stored permanantly on silica in our freezer. We screened nearly 1600 of these for their pathogencity to corn leaves and wounded corn pith tissue. We identified nine mutants that are either completely nonpathogenic to stalks and leaves of corn, or that are significantly reduced in pathogenicity.

One of the nine pathogenicity mutants has 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.

Current Research

Our current research efforts are focused on studies of the role of nitrogen and carbon sensing in establishment of biotrophic infections by C. graminicola on corn and C. higginsianum on Arabidopsis, and also on the potential roles of the mating-type regulatory genes and stress adaptation in pathogenicity of C. graminicola and G. zeae.


arrow
Expectations for Members of the Vaillancourt Lab 

Desiderata