Yuanzhong Jiang

Genome-wide identification and characterization of the Populus WRKY transcription factor family and analysis of their expression in response to biotic and abiotic stresses

Highlight text This study presents the genome-wide characterization of the Populus WRKY family under biotic and abiotic stresses. Overexpression of an SA-inducible gene, PtrWRKY89, enhanced resistance to pathogens in transgenic poplar.

Functional characterization of the poplar R2R3-MYB transcription factor PtoMYB216 involved in the regulation of lignin biosynthesis during wood formation.

Because of the importance of wood in many industrial applications, tremendous studies have been performed on wood formation, especially in lignin biosynthesis. MYB transcription factors (TFs), which consist of a large family of plant TFs, have been reported to directly regulate lignin biosynthetic genes in a number of plants. In this study, we describe the cloning and functional characterization of PtoMYB216, a cDNA isolated from Chinese white poplar (Populus tomentosa Carr.). PtoMYB216 encodes a protein belonging to the R2R3-MYB family and displays significant similarity with other MYB factors shown to regulate lignin synthesis in Arabidopsis. Gene expression profiling studies showed that PtoMYB216 mRNA is specifically expressed during secondary wall formation in wood. The 1.8-kb promoter sequence of PtoMYB216 was fused to the GUS coding sequence and introduced into wild-type A. thaliana. GUS expression was shown to be restricted to tissues undergoing secondary cell wall formation. Overexpression of PtoMYB216 specifically activated the expression of the upstream genes in the lignin biosynthetic pathway and resulted in ectopic deposition of lignin in cells that are normally unligninified. These results suggest that PtoMYB216 is specific transcriptional activators of lignin biosynthesis and involved in the regulation of wood formation in poplar.

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

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.

PtrWRKY73, a salicylic acid-inducible poplar WRKY transcription factor, is involved in disease resistance in Arabidopsis thaliana

Key messageA salicylic acid-inducible WRKY gene,PtrWRKY73,fromPopulus trichocarpa, was isolated and characterized. Overexpression ofPtrWRKY73inArabidopsis thalianaincreased resistance to biotrophic pathogens but reduced resistance against necrotrophic pathogens.AbstractWRKY transcription factors are commonly involved in plant defense responses. However, limited information is available about the roles of the WRKY genes in poplar defense. In this study, we isolated a salicylic acid (SA)-inducible WRKY gene, PtrWRKY73, from Populus trichocarpa, belonging to group I family and containing two WRKY domains, a D domain and an SP cluster. PtrWRKY73 was expressed predominantly in roots, old leaves, sprouts and stems, especially in phloem and its expression was induced in response to treatment with exogenous SA. PtrWRKY73 was localized to the nucleus of plant cells and exhibited transcriptional activation. Overexpression of PtrWRKY73 in Arabidopsis thaliana resulted in increased resistance to a virulent strain of the bacterial pathogen Pseudomonas syringae (PstDC3000), but more sensitivity to the necrotrophic fungal pathogen Botrytis cinerea. The SA-mediated defense-associated genes, such as PR1, PR2 and PAD4, were markedly up-regulated in transgenic plants overexpressing PtrWRKY73. Arabidopsis non-expressor of PR1 (NPR1) was not affected, whereas a defense-related gene PAL4 had reduced in PtrWRKY73 overexpressor plants. Together, these results indicated that PtrWRKY73 plays a positive role in plant resistance to biotrophic pathogens but a negative effect on resistance against necrotrophic pathogens.

Constitutive expression of the poplar WRKY transcription factor PtoWRKY60 enhances resistance to Dothiorella gregaria Sacc. in transgenic plants

WRKY proteins are involved in various physiological processes in plants, especially in coping with diverse biotic and abiotic stresses. However, limited information is available on the roles of specific WRKY transcription factors in poplar defense. In this study, we reported the characterization of PtoWRKY60, a Group IIa WRKY member, from Populus tomentosa Carr. The gene expression profile of PtoWRKY60 in various tissues showed that it significantly accumulated in old leaves. Phylogenetic analyses revealed that PtoWRKY60 had a close relationship with AtWRKY18, AtWRKY40 and AtWRKY60. PtoWRKY60 was induced mainly by salicylic acid (SA) and slightly by Dothiorella gregaria Sacc., jasmonic acid, wounding treatment, low temperature and salinity stresses. Overexpression of PtoWRKY60 in poplar resulted in increased resistance to D. gregaria. The defense-associated genes, such as PR5.1, PR5.2, PR5.4, PR5.5 and CPR5, were markedly up-regulated in transgenic plants overexpressing PtoWRKY60. These results indicate that PtoWRKY60 might be partly involved in the signal transduction pathway initiated by SA in Populus.

Isolation and characterization of a subgroup IIa WRKY transcription factor PtrWRKY40 from Populus trichocarpa

Salicylic acid (SA) is a defense-related key signaling molecule involved in plant immunity. In this study, a subgroup IIa WRKY gene PtrWRKY40 was isolated from Populus trichocarpa, which displayed amino acid sequence similar to Arabidopsis AtWRKY40, AtWRKY18 and AtWRKY60. PtrWRKY40 transcripts accumulated significantly in response to SA, methyl jasmonate and hemibiotrophic fungus Dothiorella gregaria Sacc. Overexpression of PtrWRKY40 in transgenic poplar conferred higher susceptibility to D. gregaria infection. This susceptibility was coupled with reduced expression of SA-associated genes (PR1.1, PR2.1, PR5.9, CPR5 and SID2) and jasmonic acid (JA)-related gene JAZ8. Decreased accumulation of endogenous SA was observed in transgenic lines overexpressing PtrWRKY40 when compared with wild-type plants. However, constitutive expression of PtrWRKY40 in Arabidopsis thaliana displayed resistance to necrotrophic fungus Botrytis cinerea, and the expression of JA-defense-related genes such as PDF1.2, VSP2 and PR3 was remarkably increased in transgenic plants upon infection with fugal pathogens. Together, our findings indicate that PtrWRKY40 plays a negative role in resistance to hemibiotrophic fungi in poplar but functions as a positive regulator of resistance toward the necrotrophic fungi in Arabidopsis.

Overexpression of Poplar PtrWRKY89 in Transgenic Arabidopsis Leads to a Reduction of Disease Resistance by Regulating Defense-Related Genes in Salicylate- and Jasmonate-Dependent Signaling

The plant hormones jasmonic acid (JA) and salicylic acid (SA) play key roles in plant defenses against pathogens and several WRKY transcription factors have been shown to have a role in SA/JA crosstalk. In a previous study, overexpression of the poplar WRKY gene PtrWRKY89 enhanced resistance to pathogens in transgenic poplars. In this study, the promoter of PtrWRKY89 (ProPtrWRKY89) was isolated and used to drive GUS reporter gene. High GUS activity was observed in old leaves of transgenic Arabidopsis containing ProPtrWRKY89-GUS construct and GUS expression was extremely induced by SA solution and SA+MeJA mixture but not by MeJA treatment. Subcellular localization and transactivation assays showed that PtrWRKY89 acted as a transcription activator in the nucleus. Constitutive expression of PtrWRKY89 in Arabidopsis resulted in more susceptible to Pseudomonas syringae and Botrytis cinerea compared to wild-type plants. Quantitative real-time PCR (qRT-PCR) analysis confirmed that marker genes of SA and JA pathways were down-regulated in transgenic Arabidopsis after pathogen inoculations. Overall, our results indicated that PtrWRKY89 modulates a cross talk in resistance to P. syringe and B. cinerea by negatively regulating both SA and JA pathways in Arabidopsis.

Isolation and Characterization of cDNAs Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase from Populus trichocarpa

Proanthocyanidins (PAs) contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA) and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are two key enzymes of the PA biosynthesis that produce the main subunits: (+)-catechin and (−)-epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05) in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus.