The L-Type Lectin-like Receptor Kinase Gene TaLecRK-IV.1 Regulates the Plant Height in Wheat

Dwarfing is important for the production of wheat (Triticumaestivum L.). In model plants, receptor-like kinases have been implicated in signal transduction, immunity, and development. However, functional roles of lectin receptor-like kinases in wheat are poorly understood. In this study, we identified an L-type lectin receptor-like kinase gene in wheat, designated as TaLecRK-IV.1, and revealed its role in plant height. Real time quantitative PCR analyses indicated that TaLecRK-IV.1 transcript level was lower in a dwarf wheat line harboring the Rht-D1b gene compared to its transcript level detected in a taller wheat line CI12633. Importantly, the virus-induced gene silencing results showed that silencing of TaLecRK-IV.1 in the wheat line CI12633 led to dwarf plants. The results of the disease resistance test performed after the gene silencing experiment suggest no significant role of TaLecRK-IV.1 in the resistance reaction of wheat line CI12633 to sharp eyespot. Gene expression analysis revealed that the transcript abundance of TaLecRK-IV.1 was more up-regulated after the exogenous application of gibberellic acid and auxin, two development-related phytohormones, compared to the gene transcript levels detected in the control plants (mock treatment). These findings support the potential implication of TaLecRK-IV.1 in the pathway controlling plant height rather than the disease resistance role, and suggest that TaLecRK-IV.1 may be a positive regulator of plant height through the gibberellic acid and auxin-signaling pathways.


Introduction
Plants possess a communication system that allows them to receive and process information. The system does not only permit them to constantly detect the changes in environmental conditions that can lead to growth and developmental modulations but also helps them in detecting external menaces such as pathogen infections. The perception and transduction of signals from environmental cues to the plant intracellular milieu are mostly governed by the receptor-like kinases [1]. The genes encoding receptor-like kinases (RLKs) are found in great numbers and are largely distributed in plant genomes. For instance, over 600 RLK genes have been identified in Arabidopsis thaliana while rice (Oryza sativa) harbors more than 1000 genes [2]. In higher plants, RLKs form a vast protein family and lectin receptor kinases (LecRKs) are a group of RLK proteins characterized by an N-terminal extracellular lectin motif [3]. Based on the variability of the N-terminal lectin domains, this category has been sub-grouped into four classes, G-, C-, L-, and LysM-types [4]. Among the four classes, the L-type is the more abundant and is characterized by the presence of three distinct domains comprising a legume lectin-like extracellular domain, a transmembrane domain, and an intracellular kinase domain [5].
In Arabidopsis, LecRK-VI.2 protein is implicated in the pattern-triggered immune responses to bacterial pathogens [6]. The lectin receptor kinase genes AtLecRK-IV.3 and signals [3]. Studies documenting the implication of lectin-receptor kinase genes on plant development are limited.
In this study, we cloned a gene in wheat encoding a LecRK-IV.1, named TaLecRK-IV.1, and found that silencing of TaLecRK-IV.1 led to dwarf plants in a wheat line CI12633. Test for disease resistance following TaLecRK-IV.1-silencing revealed no impairment of resistance of wheat line CI12633 to sharp eyespot despite the reduction of TaLecRK-IV.1 transcripts. The results suggest that TaLecRK-IV.1 is involved in the regulation of plant growth but not in disease resistance. TaLecRK-IV.1 was induced by the exogenous application of gibberellic acid (GA) or auxin. This gene may be useful in wheat breeding to develop alternative dwarf varieties without the need of employing the previous dwarfing alleles. The annotated sequence of TRIAE_CS42_4AL_TGACv1_288345_AA0945480 (https:// plants.ensembl.org/Triticum_aestivum/ (accessed on 16 February 2018)) corresponding to TaLecRK-IV.1 was identified by BLAST search using a short putative gene sequence obtained following an RNA-seq experiment. The primer pair [QF: 5 -ATACTACTGAGTTTTCTTTTG-TCC-3 and QR: 5 -TAGGGCTGTGATTCTGAGGC-3 , Supplemental Table S1] were then designed and used to amplify the predicted full length of the gene based on the database information. The amplicon from the cDNA of the wheat cultivar (cv.) CI12633 was cloned, sequenced, and analyzed; and the sequence analysis indicated the characteristic of a proteincoding gene containing an open reading frame (ORF). The deduced protein from the cloned sequence consisted of 676 amino acid residues. The amino acids sequence of TaLecRK-IV.1 protein contains a Lectin-like superfamily domain at the N-terminal region, an ATP binding site, and a protein kinase superfamily domain at the C-terminal region. Based on the classification of lectin domains, the protein belongs to the L-type (lectin-like) group, the most abundant group of lectin receptor kinases [4]. By using the amino acid sequence of TaLecRK-IV.1 as query, BLAST search in the protein database found many L-type domains containing receptor kinase IV.1 proteins from different species, such as wheat (T. aestivum), barley (Hordeum vulgare), rice (Oryza sativa), maize (Zea mays), brachypodium (Brachypodium distachyon), foxtail millet (Setaria italica), and sorghum (Sorghum bicolor). The predicted TaLecRK-IV.1protein sequence shared 61-93% identities with those of wheat, barley, rice, maize, brachypodium, foxtail millet, and sorghum (Supplemental Table S2). In Arabidopsis, the L-type LecRK proteins were classified into nine clusters and nine clades on the basis of phylogenetic relationships [4]. BLAST and phylogenetic tree analyses showed that TaLecRK-IV.1 fell into the cluster IV, clade 1, and TaLecRK-IV.1 was closer to Aegilops tauschii and Triticum aestivum L-type LecRks (Figure 1). Database accession numbers and the name of the species are shown. The phylogenetic tree was constructed using the neighbor-joining phylogeny of MEGA 5.0. TaLecRK-IV.1 Leads to Dwarf Plants CI12633, a tall wheat cultivar [25], a was employed in the VIGS experiment. Seeds were planted in pots, and seedlings at two-to three-leaf stage were inoculated with a mixture of BSMV RNA (α, β, and γ:00 or γ:TaLecRK-IV.1). Two types of mixture derived from the in-vitro transcription were set up. One contained the BSMV RNA α, β, and γ:00 and was used to inoculate CI12633 seedlings considered as control. Another mixture containing the BSMV RNA α, β, and γ:TaLecRK-IV.1 was employed to infect other CI12633 seedlings for the gene silencing purpose. About 10 days post-inoculation, symptoms of BSMV infection were observed on the newly developed leaves of more than 90% of the inoculated plants and were seen on both groups (control and TaLecRK-IV.1-silenced plants). Based on the specific PCR fragment for BSMV coat protein gene, the presence of BSMV was detected in all plants exhibiting the phenotype characteristic of BSMV infection. Importantly, the RT-qPCR analyses showed that compared to control, TaLecRK-IV.1 transcriptional level was considerably reduced in BSMV:TaLecRK-IV.1-infected wheat plants ( Figure 2). TaLecRK-IV.1 nucleotide sequence shared 92% similarity with another gene located on wheat chromosome 7AS, namely 7AS-homolog, and both were supposed to be homologous genes. The 224 bp fragment used to construct the BSMV vector to silence TaLecRK-IV.1 has 88% similarity with the corresponding fragment of the 7AS-homolog. Thus, the transcript of the 7AS-homolog was also analyzed by RT-qPCR and the result indicated that this gene was also downregulated ( Figure 2) but to a lesser extent as compared to TaLecRK-IV.1. In accordance with this result, a previous study reported that endogenous genes sharing high homology with the fragment beard by the BSMV can be silenced by VIGS, even if the inserts in the BSMV vectors are from other species [26].  Double asterisks indicate significant expression difference at 0.01 probability level as compared to the relative expression in control by using the Student's t-test.

Silencing of
Following the observation of successful BSMV infection and the gene expression levels of TaLecRK-IV.1 and its 7AS-homolog in wheat CI12633, the monitoring of the plants has permitted to realize that the plants infected with BSMV:TaLecRK-IV.1 displayed a dwarf phenotype as compared to those inoculated with BSMV:00 (control, Figure 3). In the previous experiments in our laboratory [27][28][29][30][31], dwarf phenotype was not observed when the wheat cultivar CI12633 was subjected to the VIGS experiments. Moreover, CI12633 is a tall cultivar [25], which harbors none of the well-known and largely distributed dwarfing genes (Rht-B1b and Rht-D1b). Taken togather, the results suggested that silencing of TaLecRK-IV.1 led to reduced plant height, and that TaLecRK-IV.1 might positively regulate plant height. Double asterisks indicate significant expression difference at 0.01 probability level as compared to the relative expression in control by using the Student's t-test.
Following the observation of successful BSMV infection and the gene expression levels of TaLecRK-IV.1 and its 7AS-homolog in wheat CI12633, the monitoring of the plants has permitted to realize that the plants infected with BSMV:TaLecRK-IV.1 displayed a dwarf phenotype as compared to those inoculated with BSMV:00 (control, Figure 3). In the previous experiments in our laboratory [27][28][29][30][31], dwarf phenotype was not observed when the wheat cultivar CI12633 was subjected to the VIGS experiments. Moreover, CI12633 is a tall cultivar [25], which harbors none of the well-known and largely distributed dwarfing genes (Rht-B1b and Rht-D1b). Taken togather, the results suggested that silencing of TaLecRK-IV.1 led to reduced plant height, and that TaLecRK-IV.1 might positively regulate plant height.
the relative expression in control by using the Student's t-test.
Following the observation of successful BSMV infection and the gene expression levels of TaLecRK-IV.1 and its 7AS-homolog in wheat CI12633, the monitoring of the plants has permitted to realize that the plants infected with BSMV:TaLecRK-IV.1 displayed a dwarf phenotype as compared to those inoculated with BSMV:00 (control, Figure 3). In the previous experiments in our laboratory [27][28][29][30][31], dwarf phenotype was not observed when the wheat cultivar CI12633 was subjected to the VIGS experiments. Moreover, CI12633 is a tall cultivar [25], which harbors none of the well-known and largely distributed dwarfing genes (Rht-B1b and Rht-D1b). Taken togather, the results suggested that silencing of TaLecRK-IV.1 led to reduced plant height, and that TaLecRK-IV.1 might positively regulate plant height.

The Expression of TaLecRK-IV.1 in the Wheat Line with Rht-D1b Gene
In order to hypothesize that the reduced expression of TaLecRK-IV.1 and/or its 7A-homolog gene affects plant height in wheat, we analyzed by RT-qPCR the transcript abundance of these genes in Xinong223, a wheat line harboring the Rht-D1b dwarfing gene. It was found that the expression level of TaLecRK-IV.1 was lower, while that of the 7A-homolog gene was higher in the wheat line carrying the Rht-D1b. The transcript level of TaLecRK-IV.1 was significantly lower in the dwarf wheat line compared to its expression detected in wheat cultivar CI12633, whereas an opposite expression difference (high expression) was found for the 7A-homolog gene in the Rht-D1b carrying wheat line as compared to that of the control (CI12633) (Figure 4). The results suggested that in the presence of Rht-D1b, the transcript of TaLecRK-IV.1 was not abundant, probably due to the action of the Rht allele. While the expression level of TaLecRK-IV.1 in the Rht-D1b-carrying wheat line was low, that of its 7A homologue gene was elevated in the same wheat genotype (Xinong223) as compared to the transcript abundance detected in the control (CI12633). The low expression of TaLecRK-IV.1 in a wheat line with Rht-D1b (dwarf line) was in agreement with the hypothesis that the product of this gene may be implicated in the pathway affecting plant height. These data suggested that the expression of TaLecRK-IV.1 might be influenced by the presence of the Rht-D1b, which may act upstream of TaLecRK-IV.1. Also, the data imply that when the expression of TaLecRK-IV.1 is not altered, it contributes to normal plant height, whereas its silencing leads to dwarf plants.
the control (CI12633). The low expression of TaLecRK-IV.1 in a wheat line with Rht-D1b (dwarf line) was in agreement with the hypothesis that the product of this gene may be implicated in the pathway affecting plant height. These data suggested that the expression of TaLecRK-IV.1 might be influenced by the presence of the Rht-D1b, which may act upstream of TaLecRK-IV.1.Also, the data imply that when the expression of TaLecRK-IV.1 is not altered, it contributes to normal plant height, whereas its silencing leads to dwarf plants.

Relative Expression of GA Biosynthetic Enzymes TaGA20ox and TaGA3ox in Rht-D1b-Carring Wheat Line and TaLecRK-IV.1-Silenced CI12633 Plants
To explore if the silencing of TaLecRK-IV.1 affects the expression of these enzymes, and led to dwarf phenotype, we analyzed the relative abundances of TaGA20ox and

TaLecRK-IV.1 Is Expressed in Response to Exogenous GA and Auxin Treatments
We further investigated if TaLecRK-IV.1 responds to exogenous hormone treatments. Upon GA treatment, TaLecRK-IV.1 transcript level was considerably increased from 1 to 3 Asterisks indicate significant expression difference at 0.05 (*) or 0.01 (**) probability level as compared to the relative expression in control based on the Student's t-test results.

TaLecRK-IV.1 Is Expressed in Response to Exogenous GA and Auxin Treatments
We further investigated if TaLecRK-IV.1 responds to exogenous hormone treatments. Upon GA treatment, TaLecRK-IV.1 transcript level was considerably increased from 1 to 3 h then declined at 6 h, and finally the induction resumed at 12 h before reaching the highest level at 24 h ( Figure 6A). Following exogenous auxin application, TaLecRK-IV.1 expression was first down-regulated about 30 min post-treatment. After that, the expression regained approximately to a normal level about 1 h before being up-regulated between 3 to 6 h post-treatment with auxin. After 6 h, the gene transcript declined considerably between 12 and 24 h post-treatment ( Figure 6B).  Figure 7A). From the hormone treatments, we noted that auxin-induced expression started to differentially increase when the up-regulation caused by ethylene started decreasing about 3 h post-treatment. Considering the expression trend following salicylic acid treatment, the gene was down-regulated throughout the time points except at 1 h where a somewhat increase in  Figure 7A). From the hormone treatments, we noted that auxin-induced expression started to differentially increase when the up-regulation caused by ethylene started decreasing about 3 h post-treatment. Considering the expression trend following salicylic acid treatment, the gene was down-regulated throughout the time points except at 1 h where a somewhat increase in the expression of TaLecRK-IV.1 was observed as compared to that of control ( Figure 7B). This expression trend may be explained by the fact that SA mostly induces the expression of the defense-related genes.

Resistance Responses to Sharp Eyespot of the Plants Subjected to the VIGS Experiment
As TaLecRK-IV.1 was among the differentially expressed genes (DEG) based on the RNA-seq results, the gene was predicted to contribute to the disease resistance reaction. To elucidate whether its up-regulation was an active plant defense response, TaLecRK-IV.1 was first silenced by VIGS in wheat line CI12633 and then the plants were inoculated with Rhizoctonia cerealis two weeks after BSMV infection. The results of the disease resistance test have revealed no significant difference between the level of re-

Resistance Responses to Sharp Eyespot of the Plants Subjected to the VIGS Experiment
As TaLecRK-IV.1 was among the differentially expressed genes (DEG) based on the RNA-seq results, the gene was predicted to contribute to the disease resistance reaction. To elucidate whether its up-regulation was an active plant defense response, TaLecRK-IV.1 was first silenced by VIGS in wheat line CI12633 and then the plants were inoculated with Rhizoctonia cerealis two weeks after BSMV infection. The results of the disease resistance test have revealed no significant difference between the level of resistance of the control plants (infected with BSMV:00) and those inoculated with the BSMV:TaLecRK-IV.1 vector construct (Table 1). This result suggested that TaLecRK-IV.1 may not be the major gene required in the resistance pathway to counter sharp eyespot infection. Thus, its up-regulation as revealed by RNA-seq could be related to the perturbation of the steady-state of the plants initially challenged by sharp eyespot. To confirm the location of TaLecRK-IV.1 on wheat chromosome 4A, a specific pair of primers to the gene was designed and tested on a set of DNA of Chinese Spring nullitetrasomic (N-T) lines. The PCR assay detected a single band product of expected size (~630 bp) in Chinese Spring but failed to amplified the target fragment of TaLecRK-IV.1 in nulli 4A-tetra 4B and in nulli 4A-tetra 4D (N4A-T4B & N4A-T4D, Figure 8). This result indicated that TaLecRK-IV.1 was located on wheat chromosome 4A.

TaLecRK-IV.1 Is Assigned to Chromosome 4A
To confirm the location of TaLecRK-IV.1 on wheat chromosome 4A, a specific pair o primers to the gene was designed and tested on a set of DNA of Chinese Spring nu li-tetrasomic (N-T) lines. The PCR assay detected a single band product of expected siz (~630 bp) in Chinese Spring but failed to amplified the target fragment of TaLecRK-IV.1 i nulli 4A-tetra 4B and in nulli 4A-tetra 4D (N4A-T4B & N4A-T4D, Figure 8). This resu indicated that TaLecRK-IV.1 was located on wheat chromosome 4A.

Discussion
In this study, TaLecRK-IV.1 was cloned and its expression profile was investigated The functional role of TaLecRK-IV.1 was analyzed by the mean of VIGS, and the resul showed that silencing of TaLecRK-IV.1 led to dwarf plants in CI12633. The disease infe tion test revealed no significant difference between the control plants and those subjecte to the gene-silencing experiment. This means that TaLecRK-IV.1 may be involved in th pathway regulating plant height rather than the one mounting the defense reactio against Rhizoctonia cerealis infection.
CI12633 is a tall wheat cultivar and does not carry any known dwarfing gene. In terestingly, TaLecRK-IV.1 transcript level was found to be relatively low in the dwa wheat line harboring the Rht-D1b as compared to its level in CI12633. Taken together, th low transcript level of TaLecRK-IV.1 in the Rht-D1b-harboring dwarf wheat line and th reduced height caused by its silencing support the hypothesis that this gene could pos tively regulate plant height and might act downstream of the Rht-D1b. Thus, it is hy

Discussion
In this study, TaLecRK-IV.1 was cloned and its expression profile was investigated. The functional role of TaLecRK-IV.1 was analyzed by the mean of VIGS, and the results showed that silencing of TaLecRK-IV.1 led to dwarf plants in CI12633. The disease infection test revealed no significant difference between the control plants and those subjected to the gene-silencing experiment. This means that TaLecRK-IV.1 may be involved in the pathway regulating plant height rather than the one mounting the defense reaction against Rhizoctonia cerealis infection.
CI12633 is a tall wheat cultivar and does not carry any known dwarfing gene. Interestingly, TaLecRK-IV.1 transcript level was found to be relatively low in the dwarf wheat line harboring the Rht-D1b as compared to its level in CI12633. Taken together, the low transcript level of TaLecRK-IV.1 in the Rht-D1b-harboring dwarf wheat line and the reduced height caused by its silencing support the hypothesis that this gene could positively regulate plant height and might act downstream of the Rht-D1b. Thus, it is hypothesized that in the presence of a dwarfing gene, TaLecRK-IV.1 expression might be repressed, which leads to short plant stature conferred by the action of the Rht allele, whereas in the absence of an Rht gene, TaLecRK-IV.1 may contribute to normal plant height leading to tall plants.
In Arabidopsis, lectin-like receptor kinases are believed to potentially participate in the regulation of plant growth and stress response [3]. Thus, TaLecRK-IV.1 appears to be the first reported LecRK in plant potentially involved in mediating the processes controlling plant height.
In plants, development is mainly under the control of the growth-mediated phytohormones of which the roles of auxin and gibberellins are well characterized. Here, RT-qPCR analysis of TaLecRK-IV.1 transcription revealed that the gene was induced upon GA and auxin treatments. Following the GA treatment, the gene was slightly down-regulated just after treatment ( Silencing of TaLecRK-IV.1 produces dwarf plants comparable to the phenotypic effects of the Rht genes. Thus, it was hypothesized that TaLecRK-IV.1 may be either a GA signaling gene or it acts on the GA biosynthesis pathway. To establish a relation between the role of TaLecRK-IV.1 and the GA biosynthesis, the transcript levels of TaGA20ox and TaGA3ox, two enzymes that catalyze the later steps in the GA biosynthetic pathway, were analyzed by RT-qPCR. The RT-qPCR results showed that the relative expression of TaGA20ox was higher in TaLecRK-IV.1-silenced plant than the transcript levels detected in control (CI12633), whereas the transcripts of TaGA30x was not affected by the effects of TaLecRK-IV.1-silencing. These results suggest that silencing of TaLecRK-IV.1 may cause the accumulation of TaGA20ox transcripts but without affecting the expression of TaGA3ox. This implies that TaLecRK-IV.1 may be involved in the regulation of plant height through a mechanism affecting the expression of TaGA20ox.
Increased expression of the GA oxidase genes leading to dwarfism was reported in some previous studies [32][33][34][35][36]. Ford et al. [32] reported that Rht18 semidwarfism in wheat was caused by the increased expression of the GA2oxA9 (GA 2-oxidaseA9) that led to the reduction of the bioactive GA1 content through the conversion of GA12 or GA53 into the inactive forms GA110 or GA97, respectively. Appleford et al. [34] documented that the ectopic expression of PcGA2ox1 from bean (Phaseolus coccineus) in wheat resulted in low GA content and led to strong plant height reduction but with other effects on the growth habit. Similarly, in rice, severe height reduction was observed following the overexpression of the C19 OsGA2ox1 gene [35,36], while a similar experiment overexpressing the C20 OsGA2ox6 or OsGA2ox9 yielded plants with moderate height reductions without effects on the date of anthesis or grain yields [37,38]. These studies are in accordance with the hypothesis that the accumulation of the GA oxidase enzyme is associated with a decrease in plant height. Nevertheless, the GA-responsive Rht18 is a dwarfing gene that affects the biosynthesis of GA1 through the up-regulation of TaGA2oxA9, whereas none of the gene family of the lectin receptor-like kinase was reported to be implicated in plant height control. We presume that the increased transcription of the TaGA20x detected in the TaLecRK-IV.1-silenced plants may lead to a comparable action to that of Rht18, thus preventing the accumulation of bioactive GA, and as result, leading to a height reduction. Further investigation will clarify the mechanism through which TaLecRK-IV.1 acts in the pathway controlling plant height.

Plant Materials, Growth Conditions and Treatments
Two wheat (T. aestivum) genotypes, including CI12633 and Xinong223 (a dwarf wheat variety harboring the Rht-D1b formerly known as Rht2), were used in this experiment. The dwarf wheat line was obtained from the laboratory of Professor Yin-Gang Hu (Northwest A&F University, Xianyang, China). The plant materials were grown under a 14 h light (~22 • C)/10 h dark (~10 • C) cycle in greenhouse.
Seedlings of the wheat cultivar CI12633 at three-leaf stage were sprayed with exogenous chemical precursors of gibberellic acid, auxin, ethylene, and salicylic acid. Leaf tissues corresponding to each treatment were separately collected at the following time points: 0, 0.5, 1, 3, 6, 12, and 24 h post chemicals application.

RNA Isolation and cDNA Synthesis
RNA was extracted from the collected leaf tissues using a commercial RNA isolation kit, TriZol (Invitrogen, Waltham, MA, USA). Three biological replicates were sampled from the control plants, plants used in the gene-silencing experiment, as well as from those employed in phytohormone treatment analyses. The integrity of the RNA was verified by agarose gel. Reverse transcription (RT) of RNA into cDNA was performed following a two-steps protocol using a Superscript II RT kit (TaKaRa, Kusatsu, Japan). TaLecRK-IV.1 To clone the full length of the gene TaLecRK-IV.1, a pair of primers was designed based on the putative sequence of TRIAE_CS42_4AL_TGACv1_288345_AA0945480, a predicted protein coding gene from the new reference sequence of the Chinese Spring annotated by the Earlham institute (formerly TGAC, https://plants.ensembl.org/Triticum_aestivum/ (accessed of 16 February 2018)). The PCR product amplified using the following primers ATACTACTGAGTTTTCTTTTGTCC-F and TAGGGCTGTGATTCTGAGGC-R corresponding to the expected size was cloned and then sequenced. The obtained sequence was analyzed and its open reading frame (ORF) spanning more than 2 kb was deduced. TaLecRK-IV.1 Barley Strip Mosaic Virus (BSMV) subgenome RNAs described by Holzberg et al. [26] were employed in this study to construct vectors for the silencing experiment. In the BSMV RNA, the subgenome γ was first double digested with Nhe I restriction enzyme and then used for sub-cloning with a 224 bpcDNA fragment of TaLecRK-IV.1, which was inserted in antisense orientation into the subgenome γ. The resulting recombinant γ construct was confirmed by sequencing. Subsequently, plasmids from the recombinant γ vector as well as from the BSMV α and BSMV β were isolated and linearized by restriction enzyme digestion (Mlu I for α and γ, and Spe I for β). The linearized plasmid DNAs were transcribed in vitro to RNAs following the instruction manual of RiboMAX Large Scale RNA Production-T7 kit (Promega). An equal volume (1:1:1) of RNAs derived from the transcription of linearized α, β, and γ was first mixed and then the FES buffer was added; and the mixture was used for transfecting seedlings of the wheat line CI12633 at the three-leaf stage as described by Panwar et al. [39]. After inoculation, these seedlings were immediately sprayed with RNA-free water to improve the humidity and then taken into a controlled environment where they were misted regularly for 48 h at 20~22 • C. Finally, the seedlings were kept under high humidity for another 24 h before transferring to a glass house.

Real Time PCR Analysis
RT-qPCR was performed in a 96-well plate ABI 7500 real-time PCR equipment of Applied Bio-systems. The total reaction volume was 20 µL and included 10 µL SYBR green (Premix Ex Taq II), 0.4 µL ROX Reference Dye II from TaKaRa, 0.5 µL of each primer, 4 µL of template cDNA, and 4.6 µL ddH 2 O. The reaction for each gene was replicated three times. The PCR amplification was performed according to the program defined by TaKaRa in the manual for the SYBR product. Specific primers for the genes of interest (Supplemental Table S1) were designed based on the information of the reference sequences of the target genes and after BLAST search analyses. The specificities and efficiencies of the primers were evaluated in a preliminary assay prior to the gene expression analysis. The gene expression was calculated using the relative quantification method employing the 2 −∆∆CT formula [40]. TaActin was used as an endogenous control. The quantitative PCR was employed to analyze the expression of TaLecRK-IV.1 in the plants employed in the silencing experiment and in those subjected to phytohormone treatments.

Test for Susceptibility to Sharp Eyespot
Two weeks post-BSMV infection, sterilized wheat kernels cultured to propagate a sharp eyespot and on which Rhizoctonia cerealis was well-developed were used to inoculate CI12633 wheat plants.
The fungal pathogen Rhizoctonia cerealis isolate, R0301, the most frequent and virulent in Jiansu province (China), was obtained from Jiansu Academy of Agricultural Sciences. The pathogen isolate was cultured on potato dextrose agar and then propagated on sterilized wheat kernels and toothpicks at room temperature for inoculation purposes. The plants of the wheat genotype CI12633 were inoculated at the bottom sheath. After inoculation, the relative humidity was increased to facilitate the infection of the inoculated plants. The test aimed to evaluate the resistance reaction of the plants employed in the gene-silencing experiment (i.e plants infected with BSMV:TaLecRK-IV.1 carrying a target gene insert) as compared to the control plants inoculated with BSMV:00 (which has no foreign gene fragment). Through this test of susceptibility, the involvement of the gene targeted for silencing in the resistance pathway can be inferred if its down-regulation leads to more vulnerability of the host plant following pathogen challenge. The phenotyping evaluation of the disease infection was done at 21 days post-inoculation.

Conclusions
This study suggests that the L-type lectin-like receptor kinase gene TaLecRK-IV.1 is a positive regulator of plant height and its silencing led to dwarf plants in CI12633. This gene may act downstream of the Rht alleles and play roles through the pathway governing the synthesis of bioactive GA. TaLecRK-IV.1 will be useful in wheat breeding programs as it can be targeted by the gene-editing tools for developing alternative dwarf varieties without the necessity of employing the Rht genes.