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Seminar: Evidence for a special role of the signal peptidase in the establishment of biotrophy by the maize anthracnose pathogen Colletotrichum graminicola

Lisa Vaillancourt, Ph.D., Professor of Plant Pathology, University of Kentucky
Lisa Vaillancourt

Lisa Vaillancourt

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November 18, 2019, 3:35 PM - 4:30 PM

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The fungus Colletotrichum graminicola (Ces.) Wils. causes anthracnose leaf blight and stalk rot diseases of maize. Anthracnose has been estimated to reduce potential grain yields in the U.S. by approximately 5% annually (1). Like other members of its genus, C. graminicola is hemibiotrophic (2).  Spores germinate on the plant surface and produce melanized dome-shaped appressoria, from which intracellular invasion occurs via direct penetration.  Once inside, the pathogen develops initially as a biotroph, producing thick, primary hyphae that invade living cells and are separated from the host cytoplasm by a membrane (3-6). After the invaded host cells die, the fungus switches to necrotrophic growth, producing thin secondary hyphae that are no longer surrounded by a membrane (3-6). Symptoms are produced only during the necrotrophic phase (3-7), but the biotrophic phase appears to be essential for the establishment of infection and subsequent colonization by Colletotrichum in living leaf and stalk tissues (4,8,9).  

To identify novel C. graminicola pathogenicity genes, we produced a collection of random mutants and screened them for loss of pathogenicity (10).  We identified a mutant (MT) that grew normally in culture, but caused no symptoms on maize leaves or stalks (10,11). In living maize cells, the MT germinates on the plant surface, forms appressoria, and penetrates normally. However, it fails to establish a successful biotrophic infection, and cannot colonize beyond the first invaded cell (3-6, 11). Interestingly, the MT can penetrate, colonize, and sporulate like the wild type (WT) in maize sheath cells that have been killed, e.g. by a localized application of liquid nitrogen, prior to inoculation (4).   The MT can also colonize living host cells normally if the WT is simultaneously co-inoculated nearby (4). These data suggest that the WT fungus induces accessibility of adjacent uninfected living host cells to the MT so that it may successfully establish a biotrophic infection in those cells (4).

The insertional mutation in the MT strain occurred in the 3’UTR region of a gene we called Cpr1 (Colletotrichum pathogenicity related gene 1) (11). The predicted CPR1 protein is a homolog of the Saccharomyces cerevisiae Spc3p (11). This protein is a subunit of the signal peptidase complex (SPC) located in the endoplasmic reticulum (ER) membrane. The SPC serves an essential function in all eukaryotes by processing signal peptides and targeting proteins destined for transport or secretion (12). Based on the high degree of sequence similarity to Spc3p, we hypothesize that CPR1 is a component of the fungal signal peptidase. The insertional mutation that occurred in the Cpr1 MT produced a unique conditional defect that is obvious only during early infection and biotrophic establishment in planta, and not during growth in culture, or during epiphytic or saprophytic growth in planta. Thus, this MT appears to have fortuitously revealed a previously unknown specific function for CPR1 and the signal peptidase in the induction of accessibility of living maize cells to biotrophic colonization, which is separate from its essential housekeeping function. Our current model proposes that post-translational modifications of the CPR1 protein in response to plant signals specifically regulate secretion of effector proteins that are necessary for the establishment of biotrophy. Our model addresses how a housekeeping function (processing of pre-proteins during protein transport) may have acquired specific additional roles in conditioning virulence in plant-pathogenic fungi.

References Cited:

  1. Frey, T.J., Weldekidan, T.,  Colbert, T.,  Wolters, P.J.C.C., and Hawk, J.A. (2011). Fitness evaluation of a locus that confers resistance to Colletotrichum graminicola (Ces.) GW Wils. using near-isogenic maize hybrids. Crop science 51, no. 4: 1551-1563.
  2. Crouch, J., O’Connell, R., Gan, P., Buiate, E., Torres, M.F., Beirn, L., Shirasu, K., and Vaillancourt, L. (2014). The genomics of Colletotrichum. In Genomics of plant-associated fungi: monocot pathogens, pp. 69-102. Springer Berlin Heidelberg.
  3. Mims, C.W., and Vaillancourt, L.J. (2002). Ultrastructural characterization of infection and colonization of maize leaves by Colletotrichum graminicola, and by a C. graminicola pathogenicity mutant. Phytopathology, 92: 803-812.
  4. Torres, M.F., Cuadros, D.F., and Vaillancourt, L.J. (2014). Evidence for a diffusible factor that induces susceptibility in the Colletotrichum–maize disease interaction. Molecular plant pathology, 15: 80-93.
  5. Venard, C., and Vaillancourt, L. (2007). Colonization of fiber cells by Colletotrichum graminicola in wounded maize stalks. Phytopathology, 97: 438-447.
  6. Venard, C., and Vaillancourt, L. (2007). Penetration and colonization of unwounded maize tissues by the maize anthracnose pathogen Colletotrichum graminicola and the related nonpathogen C. sublineolum. Mycologia, 99: 368-377.
  7. Bergstrom and Nicolson, 1999; Bergstrom, G. C., and Nicholson, R. L. (1999). The biology of corn anthracnose: knowledge to exploit for improved management. Plant disease 83: 596-608.
  8. Pellier, A.L., Laugé, R., Veneault-Fourrey, C., and Langin, T. (2003). CLNR1, the AREA/NIT2‐like global nitrogen regulator of the plant fungal pathogen Colletotrichum lindemuthianum is required for the infection cycle. Molecular microbiology 48, no. 3: 639-655.
  9. Stephenson, S.A., Hatfield, J., Rusu, A.G., Maclean, D.J., and Manners, J.M. (2000). CgDN3: an essential pathogenicity gene of Colletotrichum gloeosporioides necessary to avert a hypersensitive-like response in the host Stylosanthes guianensisMolecular plant-microbe interactions, 13(9), 929-941.
  10. Thon, M.R., Nuckles, E.M., and Vaillancourt, L.J. (2000). Restriction enzyme-mediated integration used to produce pathogenicity mutants of Colletotrichum graminicola. Molecular plant-microbe interactions, 13: 1356-1365.
  11. Thon, M.R., Nuckles, E.M., Takach, J.E., and Vaillancourt, L.J. (2002). CPR1: a gene encoding a putative signal peptidase that functions in pathogenicity of Colletotrichum graminicola to maize. Molecular plant-microbe interactions,15: 120-128.
  12. Dalbey, R.E., Lively, M.O., Bron, S., and Van-Dijl, J.M. (1997). The chemistry and enzymology of the type 1 signal peptidase. Protein science 6: 1129-1138.