Enhanced resistance to fungal pathogens in forest trees by genetic transformation of black spruce and hybrid poplar with a Trichoderma harzianum endochitinase gene
Introduction
Worldwide, forests constitute an invaluable resource for various ecological, social and economical aspects. For instance, forests provide recreational opportunities and wildlife habitat, while yielding firewood, pulp and paper, and lumber for human needs. Woody species, by their perennial nature, sustain various biotic stresses that increase forest decay and impair forest restoration processes [1], [2], [3]. Many fungal pathogens are responsible for this decay by causing such diseases as foliage rusts, cankers, vascular wilt diseases and root rots [2]. Spruces and poplars are important forest tree species in various regions, but they also are the target of a wide range of fungal pathogens. Armillaria root rot and Fomes root rot are two examples out of a long list of diseases affecting the root system of both spruce and poplar [4], [5], [6]. Melampsora leaf rust and Septoria leaf spot and canker are fungal pathogens that specifically affect poplar [7] while in spruce, needle rust caused by Chrysomyxa spp. and root rot caused by Cylindrocladium spp. or Fusarium spp have been described [3], [8], [9]. Several indirect measures can be taken to decrease losses caused by tree diseases. In tree nurseries, fungicides are commonly used but are expensive and often have negative effects on the environment [10]. Progress has also been made recently with integrated pest management plans that involve incorporating improved culture practices and genetic methods in addition to chemical and biological control.
DNA transformation technologies developed during the last decade were essential to identify gene functions in plant resistance processes. Genetic engineering of crop plants has also shown that genes from various origins (including plants) could be used successfully to improve pest tolerance. In parallel, continuing efforts are being made in tree genetic transformation to improve silvicultural traits (reviewed in [11], [12], [13]). Despite the fact that genetic engineering strategies have become available for tree species later than for crop plants, some laboratories have obtained transgenic trees with increased resistance to phytopathogens [14], [15], [16], [17]. Agrobacterium-mediated transformation methods are constantly improving and many research groups have published significant progress in deciduous trees such as poplar and in coniferous species such as spruce (reviewed in [11], [18]).
Previous work in plant defense—related strategies has focussed on the importance of plant chitinases in their defense mechanisms against fungal pathogens [19], [20]. These enzymes catalyze the hydrolysis of chitin, which is an important component of the fungal cell wall. Recently, plant chitinase genes were introduced into crop cultivars by genetic transformation and several species demonstrated enhanced resistance to fungal pathogens [21], [22], [23]. Moreover, detailed studies of chitinases from the biocontrol fungus Trichoderma harzianum have shown that it possess a particularly strong inhibition effect against a broad range of plant pathogens [24], [25]. An endochitinase gene from T. harzianum (ech42) was shown to encode a potent endochitinase with a stronger antifungal activity by comparison with other chitinolytic enzymes ([25] and references therein). This endochitinase gene was introduced into apple and it resulted in increased resistance to apple scab [26]. The same gene had also been introduced previously in petunia, tobacco, potato and broccoli plants resulting in increased resistance to fungal pathogens [25], [27], [28]. More recently, a similar endochitinase gene from Trichoderma virens was introduced into the cotton genome with transformants showing a high level of resistance to infection by Rhizoctonia solani and Alternaria alternata [29].
We genetically transformed black spruce and hybrid poplar with the endochitinase gene ech42 from T. harzianum and analyzed its expression in the transgenic trees. Disease resistance was evaluated following inoculation of the poplar with Melampsora medusae and of the spruce with Cylindrocladium floridanum.
Section snippets
Endochitinase construct
For plant DNA transformation, the disarmed Agrobacterium tumefaciens strains C58/pMP90 [30] and EHA [31] were used for spruce and poplar, respectively. Both Agrobacterium strains contained the binary vector pBIN19ESR [32]. This plasmid carried the complete coding sequence of the ech42 endochitinase gene from T. harzianum [33] under the control of a duplicated enhancer 35S promoter from 35SCaMV and containing the AMV leader sequence. The plasmid also carried the neomycin phosphotransferase II (
Regeneration of transgenic spruce and poplar lines
The ech42 gene from T. harzianum, under the control of a duplicated enhancer 35SCaMV promoter and containing the AMV leader sequence, was introduced via A. tumefaciens transformation into black spruce and hybrid poplar. Six potential transgenic poplar lines (Po-1, -4, -8, -11, -14, -20) and 15 potential transgenic spruce lines (Sp-A to -I, Sp-K, Sp-L, Sp-M, Sp-O, Sp-P, Sp-Q) were obtained, all of which tested positive to the presence of both the nptII and ech42 genes by PCR screening (Fig. 1(a)
Discussion
In this study, the endochitinase ech42 cDNA from the biocontrol fungus T. harzianum was successfully introduced into the genome of two woody plant species. All transgenic lines showed endochitinase expression and activity. However, despite the fact that the ech42 gene is under the control of the same promoter, the relative expression levels of the recombinant endochitinase gene are not comparable between poplar and spruce since we are dealing with two different types of plants. Endochitinase
Acknowledgements
Thanks are extended to G. Harman (Cornell University, Geneva, NY) for providing the genetic construct containing the ech42 gene. We thank B. Boyle (Canadian Forest Service, Sainte-Foy, QC) and G. Bussières (Université Laval, Sainte-Foy, QC) for their help with pathogen tests, L. Innes (ministère des Ressources naturelles et de la Faune et des Parcs, Sainte-Foy, QC) for providing the C. floridanum strain and R. Hamelin (Canadian Forest Service, Sainte-Foy, QC) for providing the M. medusae
References (45)
- et al.
Recent advances in the genetic transformation of trees
Trends Biotechnol
(2001) - et al.
Chitinases of fungi and plants: their involvement in morphogenesis and host–parasite interaction
Microbiol Rev
(1993) - et al.
Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in transgenic elite indica rice cultivars
Plant Sci
(2001) - et al.
Isolation and sequence of an endochitinase-encoding gene from a cDNA library of Trichoderma harzianum
Genetics
(1994) Health of North American forests
Science and Sustainable Development Directorate. Ottawa Canadian Forests Service
(1996)Tree disease concepts
(1991)Management of pathogens in seed orchards and nurseries
Forest Chron
(1991)- et al.
Species, diversity, and density affect tree seedling mortality from Armillaria root rot
Can J For Res
(1997) Root diseases in eastern Oregon and Washington
Northwest Sci
(2001)Heterobasidion annosum: biology, ecology, impact, and control. Wallingford, UK: CABI Publishing
(1998)
Poplar diseases
Diseases caused by cylindrocladium
Cylindrocladium root rot in Ontario bare-root nurseries: estimate of spruce seedling losses
Can J For Res
Principles of fungicide usage in container tree seedling nurseries
Tree Plant notes
In vitro culture and genetic engineering of Populus spp.: synergy for forest tree improvement
Plant Cell Tissue Organ Cult
Genetic engineering of plants to enhance resistance to fungal pathogens−a review of progress and future prospects
Can J Plant Pathol
Identification of candidate genes for use in molecular breeding—A case study with the Norway spruce defensin-like gene, Spi 1
Silvae Genet
Increased Septoria musiva resistance in transgenic hybrid poplar leaves expressing a wheat oxalate oxidase gene
Plant Mol Biol
Enhanced resistance to the poplar fungal pathogen, Septoria musiva, in hybrid poplar clones transformed with genes encoding antimicrobial peptides
Biotechnol Lett
Transgenic silver birch (Betula pendula) expressing sugarbeet chitinase 4 shows enhanced resistance to Pyrenopeziza betulicola
Plant Cell Rep
Genetic transformation of conifers and its application in forest biotechnology
Plant Cell Rep
Aggressive and defensive roles for chitinases
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