A wheat caffeic acid 3-O-methyltransferase TaCOMT-3D positively contributes to both resistance to sharp eyespot disease and stem mechanical strength

Plant caffeic acid 3-O-methyltransferase (COMT) has been implicated in the lignin biosynthetic pathway through catalyzing the multi-step methylation reactions of hydroxylated monomeric lignin precursors. However, genetic evidence for its function in plant disease resistance is poor. Sharp eyespot, caused primarily by the necrotrophic fungus Rhizoctonia cerealis, is a destructive disease in hexaploid wheat (Triticum aestivum L.). In this study, a wheat COMT gene TaCOMT-3D, is identified to be in response to R. cerealis infection through microarray-based comparative transcriptomics. The TaCOMT-3D gene is localized in the long arm of the chromosome 3D. The transcriptional level of TaCOMT-3D is higher in sharp eyespot-resistant wheat lines than in susceptible wheat lines, and is significantly elevated after R. cerealis inoculation. After R. cerealis inoculation and disease scoring, TaCOMT-3D-silenced wheat plants exhibit greater susceptibility to sharp eyespot compared to unsilenced wheat plants, whereas overexpression of TaCOMT-3D enhances resistance of the transgenic wheat lines to sharp eyespot. Moreover, overexpression of TaCOMT-3D enhances the stem mechanical strength, and lignin (particular syringyl monolignol) accumulation in the transgenic wheat lines. These results suggest that TaCOMT-3D positively contributes to both wheat resistance against sharp eyespot and stem mechanical strength possibly through promoting lignin (especially syringyl monolignol) accumulation.

Lignin is a phenolic cell wall polymer, which is composed of guaiacyl, syringyl and p-hydroxyphenyl units derived from the monolignol precursors (p-coumaryl, coniferyl, and sinapyl alcohols), respectively (Supplemental Fig. S1) [10][11][12] . Lignin is mainly deposited in the walls of certain specialized cells such as the tracheary elements, sclerenchyma and phloem fibers 13 . It plays pivotal roles in cell wall structural integrity, stem strength, water transport, mechanical support, and responses to various environmental and biotic stresses [12][13][14][15][16][17][18] . During plant-pathogen interactions, the deposition of lignin is thought to play a role as a physical barrier against infection. Lignification also can chemically modify cell walls to be more resistant to cell wall-degrading enzymes, increase the resistance of cell walls to the diffusion of toxins from the pathogens to the hosts and of nutrients from the hosts to the pathogens, produce toxic precursors and free radicals, and entrap the pathogens 11,19 . Monolignol biosynthesis genes contribute to local resistance to biotrophic and hemibiotrophic pathogens. For example, in diploid wheat (Triticum monococcum), transient knock-down of monolignol pathway enzymes [phenylalanine ammonia-lyase (PAL), caffeoyl-CoA O-methyltransferase (CCoAOMT), caffeic acid 3-O-methyltransferase (COMT) and cinnamyl alcohol dehydrogenase (CAD)] individually and pair-wise led to decreased basal immunity or penetration resistance to the fungal pathogens Blumeria graminis f. sp. tritici and Blumeria graminis f. sp. Hordei, respectively 11 . In rice (Oryza sativa L.), the cinnamoyl-CoA reductase (CCR) contributed to R-mediated immunity against the hemibiotrophic fungal pathogen Magnaporthe grisea 20,21 . In Arabidopsis and tobacco, loss or down-regulation of PAL led to decreased basal immunity to the hemibiotrophic bacterial pathogen Pseudomonas syringae 22 and to the biotrophic viral pathogen tobacco mosaic virus 23,24 , respectively. In Arabidopsis, through assessment on double mutant (cad-C cad-D), the genetic and functional analysis indicated that the CAD-C (At3g19450) and CAD-D (At4g34230) were the primary genes being involved in lignin biosynthesis 25,26 , and acted as essential components of defense response against virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv. tomato 16 . Recently, Rong et al. reported that the wheat CAD named TaCAD12 positively contributed to host defense response to a necrotrophic fungal pathogen R. cerealis 27 . More recent paper reported that a maize (Zea mays L.) CCoAOMT named ZmCCoAOMT2 was isolated 18 . ZmCCoAOMT2 has been shown to confer quantitative resistance to both southern leaf blight and gray leaf spot through altering lignin level and other metabolites of the phenylpropanoid pathway and regulation of programmed cell death 18 . However, defense role of COMT in plant species is poorly understood.
The COMT is an O-methyltransferase that can potentially act in various branches of the phenylpropanoid pathway 12,13 . In gymnosperms, the COMT is believed to catalyze caffeic acid rather than 5-hydroxyferulic acid. In angiosperms, COMT can catalyze caffeic acid and 5-hydroxyferulic acid as well flavone 13,28,29 . The highly conserved S-adenosyl methionine (SAM) binding domain in COMT proteins indicates the use of SAM as the methyl group donor to the hydroxyl group of a methyl acceptor molecule 30 . COMT catalyzes the multi-step methylation reactions of hydroxylated monomeric lignin precursors 11,12,31,32 . COMT is involved in the methylation of caffeic acid to ferulic acid, which is then hydroxylated at position five by ferulate-5-hydroxylase. The subsequent methylation by COMT at this position yields sinapic acid. Similarly, COMT catalyzes the 3-O-methylation of caffeoyl aldehyde and conifer-aldehyde/coniferyl alcohol in the process of guaiacyl and syringyl monolignol biosyntheses 11,12 . For example, Alfalfa COMT (MsCOMT) exhibits the higher catalyzing efficiency towards 5-hydroxyconiferaldehyde and caffeoyl aldehyde rather than caffeic acid 33 . In Brachypodium distachyon, COMT has high affinity for a variety of substrates including flavonoid compounds, with the greatest activity towards caffeic acid and caffeoyl aldehyde 34 . Interestingly, TaCM (GenBank accession number EF413031), a wheat COMT gene, was reported to locate on wheat chromosome 3BL and to encode an authentic COMT enzyme TaCM 2,13 . Kinetic analysis indicated that the TaCM protein exhibited the highest catalyzing efficiency towards caffeoyl aldehyde and 5-hydroxyconiferaldehyde as substrates, suggesting a pathway leading to syringyl lignin via aldehyde precursors 13 . Antisense TaCM transgenic tobacco assay indicated that the down-regulation of TaCM resulted in reduction in COMT activity and a sharp reduction in the syringl monolignol 13 . It has been demonstrated that in maize, rice and sorghum (Sorghum bicolor L.), the O-methyltransferase responsible for the production of syringyl lignin was also involved in the synthesis of lignin-linked tricin 35 . Several groups aimed to improve the production of biofuel and generated transgenic plants with deficit/reduction of syringyl lignin unit through knock-out/down of the lignin-related COMT genes in plants 13,[36][37][38][39] . However, no genetic evidence for defense roles of COMT genes has yet been reported in plants.
In this study, the probe with Agilent GeneChip number A_99_P198406, being homologous to certain plant COMTs, was identified to be in response to R. cerealis R0301 through comparative transcriptomic analysis on the sharp eyespot-resistant wheat line CI12633 and the susceptible wheat line Wenmai 6. Subsequently, this COMT gene located on wheat chromosome 3DL, designated as TaCOMT-3D, was cloned from the resistant wheat line CI12633. Importantly, through generation and evaluation of TaCOMT-3D-silencing and overexpressing wheat plants, we dissected roles of TaCOMT-3D in resistance response to R. cerealis, lignin accumulation, and stem mechanical strength in wheat. The functional characterization proved that TaCOMT-3D positively contributed to syringl monolignol biosynthesis, wheat resistance response to R. cerealis, and the stem mechanical strength.

Results
Identification, cloning, and chromosomal location of TaCOMT-3D. Microarray-based comparative transcriptomic assay was used to identify differentially expressed probes between sharp eyespot-resistant wheat line CI12633/Shanhongmai and the susceptible wheat line Wenmai 6 inoculated with R. cerealis R0301 (microarray raw data, GEO accession number GSE69245). Among them, the probe A_99_P198406, 100% matching to 3′-terminal sequence of a wheat cDNA sequence with accession number AK332908, displayed significantly transcriptional increase in sharp eyespot-resistant wheat lines (CI12633 and Shanhongmai) than in the susceptible wheat line Wenmai 6 at 4, 7, and 21 dpi (day post inoculation) with R. cerealis R0301 (Fig. 1A) complete open-reading frame (ORF), was cloned from cDNA of CI12633 stems inoculated with R. cerealis R0301 using nested PCR method and two pairs of primers designed based on the sequence of AK332908. The pairwise alignment showed that only one nucleotide difference exists between the cloned cDNA sequence of the gene from CI12633 and the corresponding sequence of AK332908. The gene promoter sequences were also cloned from CI12633 and Wenmai 6, respectively. The sequence analysis indicated that no difference existed between the gene promoter sequences from these two wheat lines (Supplemental Fig. S2).
To clarify the chromosome assignment of this gene, the cloned genomic sequence was subjected to alignment with the chromosome-based draft sequence of the common wheat (Chinese Spring) genome (http://www.wheatgenome.org/) (IWGSC). The alignment result displayed that this gene sequence shared 95.33%, 95.42%, and 100% identities with the matched sequences of the TGACv1_scaffold_195106_3AL, TGACv1_scaffold_220646_3B, and TGACv1_scaffold_249256_3DL, respectively. The data suggested that the gene should locate on the long arm of the wheat chromosome 3D. To verify this alignment on the chromosomal localization, genomic DNAs extracted from Chinese Spring Nulli-tetrasomic lines (NTs) were used as template of PCR using the gene-specific primers to confirm the chromosome location of the gene. The results proved that the gene was indeed located on the long arm of chromosome 3D (3DL) (Fig. 1B). Blast search against the nucleotide acid sequence database in the National Center for Biotechnology Information (NCBI) indicated that the gene sequence is homologous to COMT. Thus, this gene cloned from CI12633 was designated as TaCOMT-3D.

The expression profile of TaCOMT-3D.
To investigate if the gene expression is related with sharp eyespot-resistance, quantitative real-time PCR (qRT-PCR) technique was used to analyze the transcriptional levels of TaCOMT-3D in stems of five wheat lines/cultivars with different resistance degrees, including sharp eyespot-resistant lines CI12633 and Shanhongmai, moderately-resistant line Shannong 0431, moderately-susceptible wheat cultivar Yangmai 158, and susceptible cultivar Wenmai 6 at 21 dpi with R. cerealis R0301. The results showed that TaCOMT-3D transcriptional level was the highest in Shanghongmai, slightly decreased in CI12633, gradually declined in Shannong 0431 and Yangmai 158, and reached the lowest in Wenmai 6 ( Fig. 4A). The data indicated that the TaCOMT-3D transcriptional level is associated with the resistance degree of wheat against sharp eyespot caused by R. cerealis. Additionally, qRT-PCR analysis suggested that the R. cerealis biomass (represented by the expression level of R. cerealis Actin gene) increased with the pathogen inoculation duration (Fig. 4B), and the expression level of TaCOMT-3D in CI12633 was markedly elevated after infection of R. cerealis (Fig. 4C). Furthermore, the tissue expression profile of TaCOMT-3D at inflorescence stage was investigated in wheat cultivars Yangmai 16 and CI12633. As shown in Fig. 4D, at the inflorescence stage, the expression Knock-down of TaCOMT-3D suppresses host resistance to sharp eyespot. The barley stripe mosaic virus (BSMV)-based virus-induced gene silencing (VIGS) technique was used to analyze rapidly the functions of TaCOMT-3D in wheat. To knockdown TaCOMT-3D expression in the resistant wheat line CI12633 through BSMV-VIGS method, a 3′-terminal fragment (233-bp) specific to TaCOMT-3D was inserted in an antisense orientation into Nhe I restriction site of the BSMV RNAγ chain to generate BSMV:TaCOMT-3D recombinant construct (Fig. 5A). When the sharp eyespot-resistant wheat CI12633 plants had been infected with BSMV for 10 d, BSMV-infected symptom was present in both BSMV:GFP-and BSMV:TaCOMT-3D-inoculated CI12633 plants (Fig. 5B), and the expression of BSMV CP gene could be detected from stems of these plants (Fig. 5C). The results indicated that these BSMV:GFP and BSMV:TaCOMT-3D viruses successfully infected these viruses-inoculated wheat plants. qRT-PCR analysis indicated that the TaCOMT-3D transcriptional level was substantially reduced in stems of BSMV:TaCOMT-3D-infected CI12633 plants, revealing that TaCOMT-3D was successfully knock-downed in BSMV:TaCOMT-3D-infected CI12633 plants (Fig. 5D). Then, these plants were further inoculated with R. cerealis isolate WK207 to evaluate the defense role of TaCOMT-3D. At 45 dpi with R. cerealis WK207, more serious symptom of sharp eyespot was present on the stems of BSMV:TaCOMT-3D-infected CI12633 plants (infection types: 3.5, 3.2, 4.3), compared with that on the stems of BSMV:GFP-treated CI12633 plants (average infection type: 2.1) (Fig. 5E). These results proved that the silence of TaCOMT-3D in CI12633 suppressed host resistance to sharp eyespot caused by R. cerealis, suggesting that TaCOMT-3D is required for the wheat resistance against R. cerealis infection.

TaCOMT-3D overexpression improves wheat resistance to sharp eyespot. The TaCOMT-3D
overexpressing transgenic wheat lines were generated and used to further investigate the role of TaCOMT-3D in resistance response to R. cerealis. By using the primers specific to the transformation vector pWMB-TaCOMT-3D (Fig. 6A), PCR analysis results showed that the introduced TaCOMT-3D transgene was present in four wheat lines (OM1, OM2, OM3, and OM4) in T 0 -T 2 generations (Fig. 6B), suggesting that the transgene could be inheritable in these four lines. qRT-PCR analyses indicated that the transcriptional levels of TaCOMT-3D in these four transgenic lines (OM1, OM2, OM3, and OM4) were markedly elevated than in wild-type (WT) Yangmai 16 (recipient) (Fig. 6C), and the introduced TaCOMT-3D was overexpressed in these four lines. The western blotting results proved that the introduced TaCOMT-3D-His was translated into the fusion protein in these four overexpressing lines, but not in WT Yangmai 16 (Fig. 6D). The sharp eyespot severity assessments in two successive (T 1 -T 2 ) generations showed that compared with WT Yangmai 16, all these four TaCOMT-3D-overexpressing wheat lines (OM1, OM2, OM3, and OM4) displayed significantly enhanced resistance to sharp eyespot caused by R. cerealis R0301 (Fig. 6E, Table 1). For example, the average infection types and disease indices of these four TaCOMT-3D-overexpressing lines in T 2 generation were 1. 33 (Table 1). These results indicated that TaCOMT-3D overexpression improved resistance of the transgenic wheat to R. cerealis infection, and TaCOMT-3D positively participates in wheat resistance to R. cerealis infection.  if TaCOMT-3D affects the lignin biosynthesis in wheat, lignin content and composition in the second basal internode at milky stage were investigated. The examination data showed that the lignin content in TaCOMT-3D-overexpressing transgenic wheat lines was higher than that in WT Yangmai 16 plants (Table 2). Furthermore, hand-cut sections from the transgenic and WT plants were subjected to Wiesner and Mäule stains. When subjected to lignin-specific Wiesner reagent, cross sections of the TaCOMT-3D-overexpressing wheat lines displayed increased staining (red-brown) relative to the WT samples (Fig. 7A,B), which is indicative of a higher lignin level. When subjected to Mäule staining specific for syringyl monolignol, compared with WT samples, the TaCOMT-3D-overexpressing wheat lines displayed higher frequency of syringyl monolignol (Fig. 7C,D). These results suggested that TaCOMT-3D overexpression enhanced lignin biosynthesis, especially syringyl monolignol accumulation.

TaCOMT-3D overexpression enhances wheat stem mechanical strength. Previous study showed
that TaCM expression participated in lignin synthesis and consequently contributed to stem strength and the lodging-resistant trait in wheat lodging-resistant line C6001 and lodging-susceptible wheat H4564 2 . Here, to evaluate the effect of TaCOMT-3D on stem lodging resistance in wheat, the stem mechanical (breaking) strength of the second basal internode of TaCOMT-3D-overexpressing and WT wheat Yangmai 16 lines was measured using a Texture Analyser (TA) with a three-point bend test setup at milky and harvest stages, respectively. As shown in Table 3, comparing with WT Yangmai 16 plants, these four TaCOMT-3D-overexpressing wheat lines (OM1, OM2, OM3, and OM4) exhibited significantly increased mechanical strength of the second basal internode at both milky and harvest stages. These data suggested that overexpression of TaCOMT-3D enhanced the stem mechanical strength of the transgenic wheat.

Discussion
Plant COMT catalyzes the multi-step methylation reactions of hydroxylated monomeric lignin precursors, consequently is involved in the lignin biosynthesis 13 . However, little is known about COMT participating in defense responses to necrotrophic fungal phytopathogens. In this study, the wheat caffeic acid O-methyltransferase encoding gene TaCOMT-3D was identified through microarray-based comparative transcriptomic analysis. The chromosome-based draft sequence alignment and PCR analysis indicated that TaCOMT-3D located on the long arm of wheat chromosome 3D. Previously, two genetic studies reported that two QTLs on 3D locating in the intervals Xgwm61~Xgwm172-2 40 and Xgwm314~Xgwm383 41 for sharp eyespot resistance, respectively, the latter of which explained 7% of the phenotypic variation 41 . Here, qRT-PCR analysis showed that the transcriptional  level of TaCOMT-3D was higher in sharp eyespot-resistance wheat lines than in susceptible wheat lines/cultivars. To explain the differences in the expression profile of TaCOMT-3D between resistant and susceptible wheat, the gene promoter sequences were also cloned from CI12633 and Wenmai 6, respectively. However, no difference existed between the gene promoter sequences from these two wheat lines. Thus, we deduced the different     expression profile of TaCOMT-3D between resistant and susceptible wheat may be due to the differences in regulatory pathways that activate/suppress TaCOMT-3D expression in wheat. The gene expression level was obviously elevated after infection of R. ceraelis. These results imply that TaCOMT-3D may participate in defense response of wheat to R. cerealis infection. BSMV-based VIGS technique has been shown to be an effective reverse genetic tool for rapidly investigating the defense functions of interesting genes in barley and wheat [42][43][44][45][46] . In this report, the BSMV-VIGS results show that silencing of TaCOMT-3D in the resistant wheat line CI12633 significantly impairs host resistance to R. cerealis. The data suggest that TaCOMT-3D is required for resistance response of wheat, at least the resistant wheat line CI12633, to R. cerealis infection. To further confirm the defense function of TaCOMT-3D, transgenic wheat plants overexpressing TaCOMT-3D were generated and characterized. Taken molecular assay together disease resistance assessments for T 0 -T 2 generations, these results indicate that TaCOMT-3D-overexpressing transgenic wheat lines display significantly enhanced resistance to sharp eyespot during whole growth stages. Interestingly, TaCOMT-3D overexpression in wheat did not obviously affect major agronomic traits (including plant height, spike number, spike length, grain number per spike, and days to heading) in wheat (Supplemental Table S1). These results reveal that TaCOMT-3D positively contributes to host resistance to sharp eyespot caused by R. cerealis infection. To the best of our knowledge, this is the first report about a COMT that participates positively in plant resistance response to a necrotrophic fungal pathogen R. cerealis. However, the TaCOMT-3D-silencing and overexpressing wheat plants at seedling and adult stages showed typically susceptible phenotype to mixed strains (E9, E21, and E23) of Blumeria graminis f. sp. tritici, suggesting that TaCOMT-3D had no obvious effect on the wheat resistance phenotype to the mixed strains of powdery mildew. It is also important and interesting to further assay responses of TaCOMT-3D silenced and overexpressing wheat plants to infection of additional pathogens in future. Anyway, the current results broaden the current knowledge of plant defenses against pathogens. The sequence alignment of amino acid sequences showed that the TaCOMT-3D protein shares a 98.03% identity with the wheat caffeic acid 3-O-methyltransferase TaCM 13 . Sequence analyses reveal that SAM binding motif, catalytic residues, and active site substrate binding/positioning residues of TaCOMT-3D are the same as those in TaCM, even though seven amino acid differences exist between the protein sequences of TaCOMT-3D and TaCM. Interestingly, TaCOMT-3D and TaCM proteins shared 100% identity in all the biochemically functional sites, including the SAM binding motif, catalytic residues and active site substrate binding/positioning residues. The previous study showed that TaCM protein exhibited a strong catalyzing activity towards caffeoyl aldehyde, caffeoyl-CoA, 5-hydroxyferuloyl-CoA and 5-hydroxyconiferaldehyde, revealing that TaCM is a typical COMT involved in lignin (syringyl monolignol) biosynthesis 13 . Additionally, among the functional sites, 13 amino acid residues being involved in the active site substrate binding/positioning residues have only one amino acid difference between TaCOMT-3D (M123, N124, L129, A155, H159, F165, F169, M173, H176, V309, I312, M313, and N317) and the alfalfa COMT (M123, N124, L129, A155, H159, F165, F169, M173, H176, V309, I312, M313, and N317; GenBank accession no. AAB46623) 32,47 , with V instead of I in the position 312. The crystal structure of COMT in complex with S-adenosyl-L-homocysteine, ferulic acid, and 5-hydroxyconiferaldehyde provide a structural understanding of the observed substrate preferences. And these crystal structures identify residues lining the active site surface that contact the substrates 32 . These results suggest that TaCOMT-3D should possess the COMT activity and may participate in lignin (especially syringyl monolignol) biosynthesis.
In previous studies, assays of knock-out/down mutants/transgenic plants indicated that COMT participated predominantly in syringl monolignol biosynthesis and can change lignin content and composition 12,[36][37][38][39] . For example, the Brachypodium Bd5139 mutant with a single nucleotide mutation in the caffeic acid O-methyltransferase encoding gene BdCOMT6 displayed a moderately altered lignifiation in mature stems 38 . Ma and Xu reported that the expression of antisense TaCM in transgenic tobacco specifically reduced the COMT enzyme activity and marginally decreased lignin content, especially sharply reduced the syringl lignin monomer 13 . Ma reported that TaCM expression participated in lignin synthesis and was positively associated with stem strength and the lodging-resistant trait in wheat lodging-resistant line C6001 and lodging-susceptible wheat H4564 2 . However, little has been known about effect of COMT overexpression on lignin biosynthesis and stem mechanical strength in crop plants. Here, our results indicated that overexpression of TaCOMT-3D increased lignin level, especially syringyl monolignol content. Furthermore, TaCOMT-3D overexpression enhanced the basal stem mechanical strength, and the content of lignin and syringyl monolignol had a significantly positive correlation with the culm mechanical strength, which were in line with the effect of TaCM expression on lignin synthesis and stem strength in two wheat cultivars 2 . Previous studies documented that lignin accumulation and its composition (i.e., hydroxy-phenyl-, guaiacyl-, and syringyl-type monomers) are important factors influencing the breaking strength of wheat culm 2,3 . Zheng et al. showed that syringyl monolignol accumulation provided significant mechanical advantages to angiosperm species 3 . In this report, these assays suggest that TaCOMT-3D primarily enhances syringyl monolignol accumulation, leading to the increased the basal stem breaking strength. Lignin has been implicated in plant disease resistance 11,16,19 . Recently, the lignin biosynthesis-related enzyme ZmCCoAOMT2 in maize has been reported to confer quantitative resistance to southern leaf blight and gray leaf spot through increasing levels of lignin and other metabolites of the phenylpropanoid pathway 18 . Taken together, we can deduce that TaCOMT-3D enhances wheat defense response to R. cerealis and stem mechanical strength possibly through promoting lignin, especially syringyl monolignol accumulation.
In conclusion, the wheat gene TaCOMT-3D was identified through microarray-based comparative transcriptomics. The transcriptional level of TaCOMT-3D is higher in resistant wheat lines and significantly elevated after R. cerealis infection. TaCOMT-3D positively contributes to both resistance response to R. cerealis and adult stem mechanical strength possibly through promoting accumulation of lignin (especially syringyl monolignol). This study provides novel insights into the biological roles of COMT members in plants. TaCOMT-3D is a potential gene for improving resistance to both sharp eyespot and lodging. DNA or RNA extraction and cDNA synthesis. Genomic DNA for each sample was isolated from the wheat leaves using the CTAB method 48 .
Total RNA was extracted using TRIzol (Invitrogen, America), and then subjected to Rnase-free Dnase I (Promega, America) digestion and purification.
The purified RNA sample (2 µg) was reverse-transcribed to cDNA using the FastQuant RT Kit with gDNase (TransGen Biotech, China).

BSMV-VIGS assay for TaCOMT-3D function.
To generate the BSMV:TaCOMT-3D recombinant construct, a 233-bp sequence specific to TaCOMT-3D (from 1022 to 1254 nucleotides in TaCOMT-3D cDNA sequence) was sub-cloned in an antisense orientation into the Nhe I restriction site of the RNAγ of BSMV (Fig. 5A). Following the protocols described by Holzberg et al. 49 , the tripartite cDNA chains of BSMV:TaCOMT-3D or the control BSMV:GFP virus genome were separately transcribed into RNAs, mixed, and used to inoculate the leaves of wheat CI12633 plants at the two-leaf stage. Then, the plants were grown in a 14 h light (22 °C)/10 h dark (12 °C) regime. To investigate if BSMV successfully infected CI12633 plants, and to test if TaCOMT-3D transcript was down-regulated, at 10 dpi with the BSMV virus, the fourth leaves of the inoculated seedlings were collected and subjected to analysis on the transcription of the BSMV coat protein gene (CP) by RT-PCR and the transcription of TaCOMT-3D by qRT-PCR. At elongation stage, the BSMV-infected CI12633 plants were further inoculated with R. cerealis isolate WK207 mycelia according to Wei et al. 50 . The infection types (ITs) of these plants were scored at 45 dpi with R. cerealis WK207 following the protocol described by Chen et al. 7 .
TaCOMT-3D-overexpressing construct and transformation into wheat. The TaCOMT-3D ORF plus a 6 × His epitope tag sequence was sub-cloned into monocot transformation vector pWMB122 51 . In the resulting overexpression transformation vector pWMB-TaCOMT-3D (Fig. 6A), the transcript of theTaCOMT-3D-His fusion gene is driven by a maize ubiquitin (Ubi) promoter, and terminated by 3′-non-transcribed region of Agrobacterium tumefaciens nopaline synthase gene (Tnos). A total of 274 immature embryos of the wheat cultivar Yangmai 16 were transformed with through Agrobacterium harbouring the pWMB-TaCOMT-3D vector according to the protocol described by Wang et al. 52 .
Analysis of transcriptional levels of target genes. qRT-PCR analysis with specific primers TaCOMT-3D-QF (5′-AGAAGGTGCCCTCGGGT-3′) and TaCOMT-3D-QR (5′-TGCATTGGCGTA GATGTAAGTG-3′) was used to investigate the relative transcriptional levels of TaCOMT-3D in various wheat plants. qRT-PCR was performed using SYBR Green I Master Mix (TaKaRa, Japan) in a volume of 25 μl on an ABI 7500 RT-PCR system (Applied Biosystems). Reactions were set up using the following thermal cycling profile: 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s, 56 °C for 30 s, and 72 °C for 32 s. The relative transcriptional levels of the target genes was calculated using the 2 −ΔΔCT method 53 , where the wheat Actin gene TaActin was used as the internal reference. The relative transcriptional levels of the tested genes in the TaCOMT-3D-overexpressing wheat lines or in BSMV:TaCOMT-3D-infected wheat plants were relative to those in WT recipient or in BSMV:GFP-infected wheat plants. The transcriptional level of BSMV CP gene in wheat plants inoculated with BSMV, an indicator of BSMV infection, was investigated by semi-quantitative RT-PCR method with the BSMV-CP gene-specific primers (BSMV-CP-F: 5′-TGACTGCTAAGGGTGGAGGA-3′, BSMV-CP-R: 5′-CGGTTGAACATCACGAAGAGT-3′).

Western blotting analysis for TaCOMT-3D-overexpressing protein.
The TaCOMT-3D-His fusion protein in the overexpressing wheat lines was visualized via western blotting analysis. Total proteins were extracted from ~0.2 g of stems inoculated with R. cerealis R0301 for 20 d by using the tissue protein extraction kit (CWBIO, China). Total soluble proteins (~20 μg) for each line were separated on 12% SDS-PAGE and transferred to polyvinyl difluoride membranes (Amersham). The blotting membranes were incubated with 2500-fold diluted Anti-His Mouse Monoclonal Antibody (TransGen Biotech, China) at 4 °C overnight, then incubated with 4, 000-fold diluted Goat Anti-Mouse IgG (H + L), HPR conjugated secondary antibody (TransGen Biotech, China) at 22-23 °C for 1 h. The TaCOMT-3D-His fusion protein was visualized using the Pro-light HRP Chemiluminescent Kit (TransGen Biotech, China).
Assessment of response of transgenic wheat plants to R. cerealis. At the active tillering stage, at least 30 plants in T 1 and 110 plants in T 2 generations for each line of the TaCOMT-3D-overexpressing wheat lines and non-transgenic wheat Yangmai 16 (recipient) were inoculated with sterilized grains harboring the well-developed mycelia of R. cerealis isolate R0301 following the protocol of Zhu et al. 54 . According to the modified protocol described by Chen et al. 7 , at harvest stage (about 60 dpi), the infection type (IT) of each plant was scored based on the disease lesion squares 54 , and disease index (DI) for each wheat line were categorized.
Examination of stem mechanical strength. Stem strength testing of the material was carried out using a Texture Analyser (TA) with a three-point bend test setup as described by Miller et al. 55 . Briefly, the TA was fitted with a load cell with maximum loading capacity of 5 kg. The support stands were set at 2.5 cm apart (across which the 5 cm stem sample was placed) and the testing probe was set to move at a constant speed of 2 mm/s. The TA, connected to a computer, produces a real-time graphical output, representing the mechanical profile of the stem sample being tested. From this graph, F max , the absolute resistance of the stem sample to break under-load, were obtained.
Examination of lignin content and syringyl monolignol. Lignin content was quantitatively measured by using the lignin ELISA Kit (ChemFaces, China) according to the manufacturer's protocols. For histochemical analysis, fresh hand-cut sections were prepared from basal second internodes of wheat. Wiesner and Mäule staining methods were performed as previously described 26 . Briefly, Wiesner staining was performed by incubating sections in 1% phloroglucinol in ethanol: water (7:3) with 30% HCl. Mäule staining was performed by first incubating sections in KMnO 4 . After 10 min, sections were washed and acidified with HCl for 1 min, washed again, and then incubated in NaHCO 3 . Cross sections were immediately observed under an optical microscope.