Doug Dean, Lab Technician (Fall 1999, Fall 2002), M.Sc. student (2000 to 2002)


 
 
 
 
 
 

Email Doug Dean  (currently working at Children's Hospital, University of Toronto)

Abstract of poster presented at the CPS 2001 in London, Ontario. (Can. J. Plant Pathol. 23:197)

Expression of Round-Leaved Mallow (Malva pusilla) Glutathione S-Transferases Following Infection by Colletotrichum gloeosporioides f. sp. malvae.  J. D. Dean, P. H. Goodwin, T. Hsiang. Dept. of Env. Biol., Univ. Guelph, Guelph, ON

In plants, glutathione S-transferases (GST) comprise a diverse family of genes that are induced by several stimuli including herbicides, auxins, senescence and pathogen attack. However, the role of GSTs during pathogen attack has not been studied. Based on sequence homology, plant GSTs have been divided into four classes: phi, tau, zeta and theta. Four GSTs, GST199, GST287, GST442 and GST578, were identified from an EST library previously constructed from Colletotrichum gloeosporioides f. sp. malvae infected round-leaved mallow. A comparison of the deduced protein sequences to other plant GSTs indicated that GST442 and GST287 were from the tau class, GST578 was from the phi class and GST199 was from the zeta class. Relative RT-PCR analysis indicated that only GST199 was induced after infection, with a maximum level of expression at 96 hours during the necrotrophic stage. GST genes of class zeta, like GST199, are reported to be induced during the process of senescence. Relative RT-PCR of uninfected mallow that was artificially senesced by incubation in darkness showed that GST199 was induced to a lesser extent than during infection.  Therefore GST199 may play a role during pathogen attack as well as during senescence, possibly by detoxifying lipid peroxides or conjugating fungal toxins.
 
Publications

Dean JD, Goodwin PH, Hsiang T. 2005. Induction of glutathione S-transferase genes of Nicotiana benthamiana following infection by Colletotrichum destructivum and C .orbiculare and involvement of one in resistance. Journal of Experimental Botany 56:1525-1533. Four glutathione S-transferase (GST) genes, NbGSTU1, NbGSTU2, NbGSTU3 and NbGSTF1, were amplified from cDNA of Nicotiana benthamiana leaves infected with Colletotrichum destructivum using primers based on conserved regions of N. tabacum GST sequences. Expression of NbGSTU1 and NbGSTU3 increased progressively during infection by either C. destructivum or C. orbiculare, except for a slight decrease by NbGSTU1 late in the infection, whereas NbGSTU2 and NbGSTF1 expression remained relatively constant. Each of the four genes was cloned into a PVX vector for virus-induced gene silencing, and reduced expression of the four genes was detected by RT-PCR. A statistically significant increase in susceptibility of N. benthamiana to infection following gene silencing was found only for NbGSTU1-silenced plants, which had 130% more lesions and 67% more colonization by C. orbiculare compared to control plants. These results demonstrate that that different GST genes respond in different ways to fungal infection, and at least one plant GST gene has an important role in disease development. PDF Reprint

Dean, J. Doug, Paul H. Goodwin and Tom Hsiang. 2003. Colletotrichum gloeosporioides infection induces differential expression of glutathione S-transferase genes in Malva pusilla. Functional Plant Biology 30:821-828. Among a collection of 840 expressed sequence tags of Malva pusilla leaves infected with Colletotrichum gloeosporioides f. sp. malvae (Cgm), a total of four different glutathione S-transferase (GST) (EC 2.5.1.18) genes were identified, each showing a different pattern of expression following infection. MpGSTU1 and MpGSTU2 were members of the class tau GSTs, MpGSTF1 was a member of the class phi GSTs, and MpGSTZ1 belonged to the class zeta GSTs. Infection by Cgm occurs by a hemibiotrophic process with an initial biotrophic phase preceding the necrotrophic phase and the appearance of symptoms. Expression of MpGSTZ1 progressively increased during infection, corresponding directly with the growth of the pathogen. Expression of MpGSTU2 was similar to that of MpGSTZ1, except for a greater increase during the late necrotrophic phase. MpGSTU1 expression remained relatively constant throughout the infection, whereas MpGSTF1 expression was induced primarily during the conversion from the biotrophic to necrotrophic phases of infection. Incubation of healthy mallow leaves in the dark resulted in decreased expression of MpGSTF1 and MpGSTU1, but not MpGSTZ1 and MpGSTU2. The differential expression patterns indicate that these mallow GST genes play a variety of roles in healthy and fungal-infected leaf tissue.  PDF Reprint
 

Dean, J.D, P.H. Goodwin, and T. Hsiang. 2002. Comparison of relative RT-PCR and northern blot analyses to measure expression of b-1,3-Glucanase in Nicotiana benthamiana infected with Colletotrichum destructivum. Plant Molecular Biology Reporter 20:347-356. Although northern blot analysis is effective for quantifying gene expression, reverse transcription-polymerase chain reaction (RT-PCR) is much more sensitive. Obtaining quantitative RT-PCR results, however, can be challenging. Relative RT-PCR uses standard PCR techniques but permits the comparison of transcript quantities between samples by coamplifying the gene of interest with a housekeeping gene that acts as an internal control. To analyze the expression of a plant gene encoding a pathogenesis-related protein, such as b-1,3-glucanase, a translation elongation factor 1a (EF-1a) gene was selected as an internal control. Northern blot analysis demonstrated constitutive expression of the plant EF-1a gene following infection of Nicotiana benthamiana by Colletotrichum destructivum. Primers for the gene of interest and internal control were compatible, and 35 cycles of amplification gave reproducible relative RT-PCR results for b-1,3-glucanase gene expression. A high correlation was observed between the relative quantification of b-1,3-glucanase gene expression as determined by northern blot and relative RT-PCR analyses, demonstrating the validity of relative RT-PCR with a plant EF-1a gene as a control.   PDF Reprint

Hsiang, T. and J.D. Dean. 2001. DNA sequencing for anastomosis grouping of Rhizoctonia solani isolates from Poa annua. International Turfgrass Society Research Journal 9:674- 678.  The internal transcribed spacer region of the ribosomal DNA from four isolates of Rhizoctonia solani Kuehn from Poa annua L. and one from Agrostis palustris Huds. were sequenced, and compared to sequences from other R. solani isolates representative of 12 anastomosis groups (AG), with multiple sequences from AG1, AG2-1, AG2-2 and AG4.  The sequence alignment of the 789 bp region was subjected to distance and parsimony analyses.  Both analyses showed that representatives of the different anastomosis groups clustered separately, although branching patterns were not exactly the same in the two dendrograms.  Isolates from P. annua all clustered with AG4 HGII. An isolate from A. palustris in Ontario clustered with AG2-1, while an isolate from the same host in Wisconsin clustered with AG1-IB.  Screening of a larger number of turfgrass isolates from a wider geographic collection may reveal whether P. annua is always associated with AG4 HGII, and the group or subgroup with which A. palustris is most frequently associated.  PDF Reprint