Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa, and its constitutive expression enhances fungal resistance in transgenic plants

Abstract

The flavonoid-derived proanthocyanidins (PAs) are one class of the major defence phenolics in poplar leaves. Transcriptional activation of PA biosynthetic genes, resulting in PA accumulation in leaves, was detected following infection by the fungal Marssonina brunnea f.sp. multigermtubi using digital gene expression analysis. In order to study PA biosynthesis and its induction by fungi, a putative leucoanthocyanidin reductase gene, PtrLAR3, was isolated from Populus trichocarpa. Sequence comparison of PtrLAR3 with other known leucoanthocyanidin reductase proteins revealed high amino acid sequence similarity. Semi-quantitative reverse-transcription (RT) PCR and quantitative real-time PCR analysis demonstrated that PtrLAR3 was expressed in various tissues and the highest level of expression was observed in roots. Overexpression of PtrLAR3 in Chinese white poplar (Populus tomentosa Carr.) led to a significant plant-wide increase in PA levels. In vitro assays showed that crude leaf extracts from 35S:PtrLAR3 transformants were able to inhibit significantly the hyphal growth of M. brunnea f.sp. multigermtubi compared to the extracts from control plants. The transgenic 35S:PtrLAR3 poplar plants displayed a significant (P < 0.05) reduction in their disease symptoms compared with the control. RT-PCR analysis showed that PtrLAR3 expression was up-regulated in all transformants. These results suggested that constitutive expression of endogenous PtrLAR3 could be exploited to improve resistance to fungal pathogens in poplar.

Fig. 1.

Quantitative analysis of changes in transcript levels of flavonoid pathway genes in response to infection of poplar leaves with Marssonina brunnea f.sp. multigermtubi using digital gene expression analysis.

Fig. 2.

Levels of proanthocyanidins (PAs) in Populus tomentosa Carr. (A) Soluble PAs in different tissues from mature plants. (B) Insoluble PAs in different tissues from mature plants. (C) Soluble PAs in leaves at 0, 2, 4, 6, and 8 days after inoculation with Marssonina brunnea f.sp. multigermtubi. (D) Insoluble PAs in leaves at 0, 2, 4, 6, and 8 days after inoculation with M. brunnea f.sp. multigermtubi. Soluble PAs were determined by reaction with dimethylaminocinnamaldehyde reagent; insoluble PAs were determined by butanol/HCl hydrolysis and estimation of resulting anthocyanidin. Asterisks indicate significant differences using Student’s t-test (P < 0.05).

Fig. 3.

Comparison of PtrLAR3-deduced amino acid sequence with other LAR proteins. (A) Sequence alignment of the deduced PtrLAR3 sequence with other LAR sequences. Sequences are from Desmodium uncinatum (DuLAR, CAD79341), Lotus uliginosus (LuLAR, AAU45392), Populus tomentosa (PtrLAR3, EEF06163), Vitis vinifera (VvLAR1, AAZ82410; VvLAR2, AAZ82411). Identical amino acids are indicated by black letters on a white background, conservative amino acids by black on a dark gray background, and similar amino acids by black on a light gray background. Asterisks indicate the RFLP, ICCN, and THD motifs. Peptide sequences were aligned with the DNAMAN program. (B) Phylogenetic relationships of LAR proteins from P. tomentosa Carr. and selected species. Phylogenetic analysis was performed by the neighbour-joining method using MEGA version 4. Bar , 0.05 substitutions per site. GenBank accession numbers are: GrLAR1 (Gossypium raimondii, CAI56319); GrLAR2 (G. raimondii, CAI56325); HvLAR (Hordeum vulgare, CAI56320); LcLAR1 (Lotus corniculatus, ABC71326); LcLAR2 (L. corniculatus, ABC71328); LuLAR (Lotus uliginosus, AAU45392); MdLAR1 (Malus × domestica, AAX12185); MdLAR2 (Malus × domestica, AAX12186); MtLAR (Medicago truncatula, CAI56327); OsLAR (Oryza sativa, CAI56328); PcLAR (Phaseolus coccineus, CAI56322); PeLAR (Pinus taeda, CAI56321); PsLAR1 (Pyrus communis, ABB77696); PsLAR2 (Pyrus communis, ABB77697); PtrLAR1 (P. tomentosa, EEE89746); PtrLAR2 (P. tomentosa, EEF01056); PtrLAR3 (P. tomentosa, EEF06163); TcLAR (Theobroma cacao, ADD51358).

Fig. 4.

PtrLAR3 expression in Populus trichocarpa tissues. (A) Semi-quantitative RT-PCR analysis of PtrLAR3 expression in various tissues of P. trichocarpa. (B) Quantitative real-time PCR analysis of PtrLAR3 transcript levels in various tissues of P. trichocarpa. Poplar 18S expression was used as a control. Total RNA was isolated from roots (R), stems (S), petioles (P), mature leaves (ML), and young leaves (YL).

Fig. 5.

PA accumulation in different tissues of transgenic lines constitutively expressing PtrLAR3. PAs were localized by staining different tissues of control and PtrLAR3-overexpressor plants with the PA-specific stain dimethylaminocinnamaldehyde (DMACA; blue). (A–D) Control stem, leaf, petiole, and root, respectively. (E–H) PtrLAR3-overexpressing stem, leaf, petiole, and root, respectively. (I) Soluble PA levels in different tissues as determined by extraction and reaction with DMACA reagent. (J) Insoluble PA levels in different tissues. CK, empty-vector control; 2, 5, and 6, PtrLAR3-overexpressing lines. Asterisks indicate significant differences using Student’s t-test (P < 0.05).

Fig. 6.

In vitro antifungal activity of crude extracts from transgenic 35S:PtrLAR3 plants. (A, B) Inhibition of hyphal growth of Marssonina brunnea f.sp. multigermtubi on the plate supplied with crude leaf extracts from control plants (A) and PtrLAR3-overexpressing plants (B). (C, D) Microscopic observation of hyphal growth of M. brunnea f.sp. multigermtubi on plates without (C) and with (B) crude leaf extracts. Photomicrographs were taken after 72 h of incubation of M. brunnea f.sp. multigermtubi; bars, 100 μm. (E) Quantitative measurement of the inhibition of fungal growth of M. brunnea f.sp. multigermtubi with crude leaf extracts from control and PtrLAR3-overexpressing plants. CK, empty-vector control; 2, 5, and 6, PtrLAR3-overexpressing lines. Values are means of at least three replications. Error bars indicate standard deviations.

Fig. 7.

Resistance of transgenic poplar plants inoculated with Marssonina brunnea f.sp. multigermtubi. Poplar leaves infected with M. brunnea f.sp. multigermtubi were photographed 4 days after inoculation. (A) Empty-vector control leaves. (B) Transgenic leaves from 35S:PtrLAR3 line 2. (C) Mean infected area of transgenic lines to the fungal pathogen; PtrLAR3 confers resistance to M. brunnea f.sp. multigermtubi in transgenic poplar plants. (D) Semi-quantitative reverse-transcription PCR analysis of PtrLAR3 expression in leaves of transgenic poplar plants; ethidium bromide-stained products amplified with PtrLAR3-specific primers from transgenic poplar cDNA and control cDNA. (E) Quantitative real-time PCR analysis of PtrLAR3 transcript levels in leaves of transgenic poplar plants. CK, empty-vector control; 2, 5, and 6, PtrLAR3-overexpressing lines. Values are means of three replications. Error bars indicate standard deviation. Asterisks indicate a statistically significant difference between control and transgenic plants (P < 0.05 by Student’s t-test).