Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens

Abstract

Disease resistance in transgenic plants has been improved, for the first time, by the insertion of a gene from a biocontrol fungus. The gene encoding a strongly antifungal endochitinase from the mycoparasitic fungus Trichoderma harzianum was transferred to tobacco and potato. High expression levels of the fungal gene were obtained in different plant tissues, which had no visible effect on plant growth and development. Substantial differences in endochitinase activity were detected among transformants. Selected transgenic lines were highly tolerant or completely resistant to the foliar pathogens Alternaria alternata, A. solani, Botrytis cinerea, and the soilborne pathogen Rhizoctonia solani. The high level and the broad spectrum of resistance obtained with a single chitinase gene from Trichoderma overcome the limited efficacy of transgenic expression in plants of chitinase genes from plants and bacteria. These results demonstrate a rich source of genes from biocontrol fungi that can be used to control diseases in plants.

Figure 1

Cassettes containing ThEn42 (genomic or cDNA) used for plant transformation. (A) CaMV35S-Pro = promoter; nopaline synthase (NOS) = transcription terminator; coding sequence for the fungal signal peptide (FSP), plant secretion peptide (PSP), fungal putative activation peptide (AP). (B) The 5′ noncoding region of p35S-psCHIT42 with PSP (pTE 28.1) and p35S-CHIT42 with FSP. Box indicates mRNA stabilization consensus sequence, and topline indicates A+T rich regions (20). Bold letters indicate consensus sequences for transcription in the ATG region compared with TAA ACA ATG GCT (33). (C) Elements reducing mRNA stability at the 3′ noncoding region of the fungal gene (33) are boxed or underlined. In p35S-psCHIT42, the region downstream from restriction site SspI (arrowhead) was removed.

Figure 2

Analyses of representative transgenic (T) tobacco and potato lines. C, control plants (nontransformed or empty-vector transformed): Cs, tobacco cv. Samsun NN; Cx, tobacco cv. Xhantii; Cp, potato cv. Desiree. Representative samples of transgenic lines are shown: Ts(no. of plant line) = tobacco cv. Samsun NN transformed with p35S-ThEn42; Tx (no. of plant line) = tobacco cv. Xhantii transformed with p35S-CHIT42 (Tx1) or with p35S-psCHIT42 (Tx2 and Tx3); Tp(no. of plant line) = potato cv. Desiree transformed with p35S-ThEn42; E = pure endochitinase from T. harzianum culture. (A) PCR amplification of the fungal transgene using transgenic, control plant DNA or control fungal DNA (F) with nested primer sets EC1-Bio1N and EC1-BB2 producing 250- and 800-bp bands, respectively (indicated by arrows) (18). (B) Northern analysis of plant tissues using the ThEn-42 cDNA sequence as a probe and visualization of bands after 3–5 days of exposure. (C) Southern analysis of BamHI-digested tobacco and potato DNA probed with an EcoRI 1.7-kb fragment of ThEn-42. (D) Western analysis of soluble leaf proteins of tobacco and potato separated by SDS/PAGE and visualized with anti-endochitinase antibodies and a standard alkaline phosphatase immunoassay.

Figure 3

Gold immunolabeling of transgenic tobacco cv. Samsun NN (Ts1) transformed with p35S-ThEn42. Cy, cytoplasm; Exc, extracellular space.

Figure 4

Resistance of Trichoderma endochitinase tobacco plants to A. alternata. (A) Leaves of transgenic (line Ts3, on left) and control (on right) tobacco; dark dots indicate inoculation points. (B) Disease symptoms 11 days after inoculation and endochitinase activity in leaf protein extracts of representative transgenic lines (see legend to Fig. 2); control is an average of different lines of Cs and Cx; endochitinase activity was expressed as pmol of 4-methylumbelliferone released from 4-methylumbelliferyl-β-d-N-N′-N"-triacetylchitotriose/min/μg of protein (–31).

Figure 5

Resistance of Trichoderma endochitinase tobacco plants to B. cinerea and of potato plants to A. solani. (A) Size of lesions (mm²) produced on leaves of different tobacco transgenic lines (see legend to Fig. 2) and controls (average of Cs and Cx lines) 10 and 14 days after inoculation with B. cinerea agar plugs. (B) Number of lesions observed on different potato transgenic lines and control (see legend to Fig. 2) 9 days after spray inoculation with A. solani.

Figure 6

Resistance of Trichoderma endochitinase tobacco plants to R. solani. (A) Seedlings grown 5 days on water agar containing R. solani: controls (Cs) without pathogen (1) or with pathogen (2); transgenics (Ts3) without pathogen (3) or with pathogen (4). (B) Transgenic and control plants grown on soil infested with R. solani; best standing plants are transgenic (boxes on right and asterisks) compared with plants grown on noninfected soil (left corner).

Figure 7

Resistance of Trichoderma endochitinase tobacco plants to R. solani. Plant growth from 14 to 30 days on R. solani-infested soil relative to growth on noninfested soil (expressed as a percentage of plant height obtained on noninfested soil, which was taken as 100%). Control plants (indicated by arrows) and representative transgenic lines (see legend to Fig. 2); Tx (2 to 10 and 14) transformed with p35S-psCHIT42; Tx (1, 11, 18, 21, 23, and 31) transformed with p35S-CHIT42.

Figure 8

Resistance of Trichoderma endochitinase potato plants to R. solani. Percent survival of control and representative transgenic lines (see legend to Fig. 2) grown on R. solani-infested soil measured at different incubation periods.