Stripe Mosaic Virus-induced Gene Silencing Research Articles

A newly identified photosystem II Subunit P gene TaPsbP4A-1 in Triticeae species negatively regulates wheat powdery mildew resistance

The photosystem II (PSII) Subunit P (PsbP) protein is a component of its oxygen-evolving complex, which can oxidize water to produce oxygen using light energy and is critical to the core components and stability of PSII. Using the whole-genome information, the PsbP genes of 10 plant species were comprehensively identified. The expression patterns of wheat PsbPs under Blumeria graminis f. sp. tritici (Bgt) infection were assessed using qRT-PCR, and the functions of TaPsbPs in wheat powdery mildew resistance were studied using barley stripe mosaic virus-induced gene silencing. In total, 122 PsbP genes were divided into 8 classes with similar gene structures. No tandem repeat events were identified in wheat PsbP, suggesting that the PsbP genes in common wheat were donated by its diploid progenitor species. The expression levels of TaPsbP2A-1, TaPsbP3A-1, TaPsbP4A-1, TaPsbP4A-2, and TaPsbP7A-2 were induced by Bgt. The silencing of TaPsbP4A-1 increased the resistance of common wheat ‘Bainong AK58’ to Bgt. This study provides valuable information for functional and evolutionary research on the PsbP gene family.

JAZ1 gene regulates starch biosynthesis and changes physicochemical properties in wheat grains

Starch is the main component of crop grains. Jasmonate-ZIM domain (JAZ1), is a transcriptional repressor in the jasmonate signaling pathway, has been widely studied in plant development, and stresses responses. However, its function in crop starch biosynthesis remains unclear. In this study, the intrinsic relationship between TaJAZ1 and starch biosynthesis was studied using barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) method. The results showed that, compared with the control, grain length, thousand kernel weight, contents of total starch and amylopectin were significantly increased in grains of BSMV-TaJAZ1-silenced plants. And its starch ultrastructures, such as volume, surface area, relative crystallinity, and some viscosity parameters were also significantly changed in above grains. Moreover, the molecular mechanisms of TaJAZ1 involving starch biosynthesis were further explored by measuring the expression levels of starch synthesis-related genes. Results showed that TaJAZ1 gene silencing increased the expression of TaAGPS1, TaAGPL1, TaBEIIb and TaSSI and decreased TaGBSSI or TaSSIIIa, while their expressions were separately decreased or increased in TaJAZ1 transiently overexpression wheat plants. To conclude, these findings showed that TaJAZ1 involved starch synthesis by affecting the expression levels of six starch synthesis enzyme-related genes further changing the components, ultrastructures, and physicochemical properties of starch in wheat grain.

TaAP2-10, an AP2/ERF transcription factor, contributes to wheat resistance against stripe rust

The AP2/ERF TF (transcription factor) family is involved in regulating plant responses to various biotic and abiotic stresses. Nevertheless, understanding of the function of AP2/ERF TFs in wheat (Triticum aestivum L.) resistance against the obligate biotrophic stripe rust fungus (Puccinia striiformis f. sp tritici, Pst) remains limited. From a wheat–Pst incompatible interaction cDNA library, the transcript of TaAP2-10 was identified to be significantly induced during Pst infection. TaAP2-10, encodes an AP2 TF with two typical AP2-binding domains. There are three homologues of TaAP2-10 in the wheat genome, located on chromosome 6A, 6B and 6D. TaAP2-10 is localized in the nucleus of wheat protoplasts. A transactivation assay in yeast revealed that TaAP2-10 had transcriptional activation activity that was dependent on its C-terminal region. Quantitative real-time PCR (qRT-PCR) analyses verified that the expression of TaAP2-10 was specifically upregulated by avirulent Pst infection but not by virulent Pst, suggesting its role in wheat resistance to Pst. Furthermore, TaAP2-10 is also induced by abiotic stresses and hormone treatments, particularly under PEG4000 and abscisic acid (ABA) treatments, indicating its potential role in facilitating wheat adaptation to environmental stresses. Silencing TaAP2-10 by barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) significantly reduced wheat resistance against Pst, resulting in a decreased reactive oxygen species (ROS) burst, and promoted Pst growth and development. These findings suggest that TaAP2-10, as a nuclear-localized transcription factor, positively regulates wheat resistance to Pst.

Two functional CC-NBS-LRR proteins from rye chromosome 6RS confer differential age-related powdery mildew resistance to wheat.

Rye (Secale cereale), a valuable relative of wheat, contains abundant powdery mildew resistance (Pm) genes. Using physical mapping, transcriptome sequencing, barley stripe mosaic virus-induced gene silencing, ethyl methane sulfonate mutagenesis, and stable transformation, we isolated and validated two coiled-coil, nucleotide-binding site and leucine-rich repeat (CC-NBS-LRR) alleles, PmTR1 and PmTR3, located on rye chromosome 6RS from different triticale lines. PmTR1 confers age-related resistance starting from the three-leaf stage, whereas its allele, PmTR3, confers typical all-stage resistance, which may be associated with their differential gene expression patterns. Overexpression in Nicotiana benthamiana showed that the CC, CC-NBS, and CC-LRR fragments of PMTR1 induce cell death, whereas in PMTR3 the CC and full-length fragments perform this function. Luciferase complementation imaging and pull-down assays revealed distinct interaction activities between the CC and NBS fragments. Our study elucidates two novel rye-derived Pm genes and their derivative germplasm resources and provides novel insights into the mechanism of age-related resistance, which can aid the improvement of resistance against wheat powdery mildew.

The Leucine-Rich Repeat Receptor-Like Kinase Protein TaSERK1 Positively Regulates High-Temperature Seedling Plant Resistance to Puccinia striiformis f. sp. tritici by Interacting with TaDJA7.

Somatic embryogenesis receptor kinases (SERKs) belong to the leucine-rich repeat receptor-like kinase (LRR-RLK) subfamily, and many LRR-RLKs have been proven to play a key role in plant immune signal transmission. However, the functions of SERKs in resistance to stripe rust caused by Puccinia striiformis f. sp. tritici remains unknown. Here, we identified a gene, TaSERK1, from Xiaoyan 6, a wheat cultivar possessing high-temperature seedling-plant (HTSP) resistance to the fungal pathogen P. striiformis f. sp. tritici and expresses its resistance at the seedling stage. The expression level of TaSERK1 was upregulated upon P. striiformis f. sp. tritici inoculation under relatively high temperatures. The transcriptional level of TaSERK1 was significantly increased under exogenous salicylic acid and brassinosteroids treatments. The barley stripe mosaic virus-induced gene silencing assay indicated that TaSERK1 positively regulated the HTSP resistance to stripe rust. The transient expression of TaSERK1 in tobacco leaves confirmed its subcellular localization on the plasma membrane. Furthermore, TaSERK1 interacted with and phosphorylated the chaperone protein TaDJA7, which belongs to the heat shock protein 40 subfamily. Silencing TaDJA7 compromised the HTSP resistance to stripe rust. The results indicated that when the membrane immune receptor TaSERK1 perceives the P. striiformis f. sp. tritici infection under relatively high temperatures, it transmits the signal to TaDJA7 to activate HTSP resistance to the pathogen.

Transcriptome profiling uncovers the lncRNA-mediated regulatory networks associated with tolerance to cadmium stress in barley

Cadmium (Cd) is one of the most harmful and widespread toxic heavy metal contaminants. Long non-coding RNAs (lncRNAs) play vital roles in response to abiotic stress by participating in the gene regulatory network. Here, we performed a transcriptome analysis of lncRNAs in roots of the two contrasting barley genotypes, namely Zhenong8 (ZN, Cd-tolerant) and W6nk2 (Cd-sensitive). In total, 9937 novel lncRNAs were identified, and 5758 lncRNA-target pairs were predicted to be cis-acting and 4159 to be trans-acting. Further comparison of genotypic differences revealed 195 novel lncRNAs as differentially expressed between the two genotypes in response to Cd stress. The miRanda prediction and expression relationship analyses among miRNA, lncRNA, and mRNA revealed that 8 lncRNAs may act as target mimics of miRNAs in barley. Among them, HvGAMYB was predicted to be the target gene of miR319a_1 and miR319b-3p, with LTCONS_00177709 and LTCONS_00185175 acting as endogenous competitors. Barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) of HvGAMYB in ZN8 led to hypersensitivity to Cd stress with significantly increased Cd concentration in both roots and shoots as well as Cd accumulation in shoots. Moreover, silencing HvGAMYB suppressed photosynthesis and antioxidant-related genes but stimulated the production of reactive oxygen species (ROS) after Cd exposure, leading to exacerbated Cd-induced MDA accumulation and cell death. Our findings provide new knowledge and insights into the regulatory functions of lncRNAs in plant Cd tolerance.

PM2b, a CC-NBS-LRR protein, interacts with TaWRKY76-D to regulate powdery mildew resistance in common wheat.

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a destructive disease of wheat throughout the world. Host resistance is considered the most sustainable way to control this disease. Powdery mildew resistance gene Pm2b was mapped to the same genetic interval with Pm2a and PmCH1357 cloned previously, but showed different resistance spectra from them, indicating that they might be caused by different resistance genes or alleles. In this study, Pm2b was delimited to a 1.64 Mb physical interval using a large segregating population containing 4,354 F2:3 families of resistant parent KM2939 and susceptible cultivar Shimai 15. In this interval, TraesCS5D03G0111700 encoding the coiled-coil nucleotide-binding site leucine-rich repeat protein (CC-NBS-LRR) was determined as the candidate gene of Pm2b. Silencing by barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) technology and two independent mutants analysis in KM2939 confirmed the candidate gene TraesCS5D03G0111700 was Pm2b. The sequence of Pm2b was consistent with Pm2a/PmCH1357. Subcellular localization showed Pm2b was located on the cell nucleus and plasma membrane. Pm2b had the highest expression level in leaves and was rapidly up-regulated after inoculating with Bgt isolate E09. The yeast two-hybrid (Y2H) and luciferase complementation imaging assays (LCI) showed that PM2b could self-associate through the NB domain. Notably, we identified PM2b interacting with the transcription factor TaWRKY76-D, which depended on the NB domain of PM2b and WRKY domain of TaWRKY76-D. TaWRKY76-D negatively regulated the resistance to powdery mildew in wheat. The specific KASP marker K529 could take the advantage of high-throughput and high-efficiency for detecting Pm2b and be useful in molecular marker assisted-selection breeding. In conclusion, cloning and disease resistance mechanism analysis of Pm2b provided an example to emphasize a need of the molecular isolation of resistance genes, which has implications in marker assisted wheat breeding.

A transcriptomic-guided strategy used in identification of a wheat rust pathogen target and modification of the target enhanced host resistance to rust pathogens.

Transcriptional reprogramming is an essential feature of plant immunity and is governed by transcription factors (TFs) and co-regulatory proteins associated with discrete transcriptional complexes. On the other hand, effector proteins from pathogens have been shown to hijack these vast repertoires of plant TFs. Our current knowledge of host genes' role (including TFs) involved in pathogen colonization is based on research employing model plants such as Arabidopsis and rice with minimal efforts in wheat rust interactions. In this study, we begun the research by identifying wheat genes that benefit rust pathogens during infection and editing those genes to provide wheat with passive resistance to rust. We identified the wheat MYC4 transcription factor (TF) located on chromosome 1B (TaMYC4-1B) as a rust pathogen target. The gene was upregulated only in susceptible lines in the presence of the pathogens. Down-regulation of TaMYC4-1B using barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) in the susceptible cultivar Chinese Spring enhanced its resistance to the stem rust pathogen. Knockout of the TaMYC4-1BL in Cadenza rendered new resistance to races of stem, leaf, and stripe rust pathogens. We developed new germplasm in wheat via modifications of the wheat TaMYC4−1BL transcription factor.

Identification of ankyrin-transmembrane-type subfamily genes in Triticeae species reveals TaANKTM2A-5 regulates powdery mildew resistance in wheat.

The ankyrin-transmembrane (ANKTM) subfamily is the most abundant subgroup of the ANK superfamily, with critical roles in pathogen defense. However, the function of ANKTM proteins in wheat immunity remains largely unexplored. Here, a total of 381 ANKTMs were identified from five Triticeae species and Arabidopsis, constituting five classes. Among them, class a only contains proteins from Triticeae species and the number of ANKTM in class a of wheat is significantly larger than expected, even after consideration of the ploidy level. Tandem duplication analysis of ANKTM indicates that Triticum urartu, Triticum dicoccoides and wheat all had experienced tandem duplication events which in wheat-produced ANKTM genes all clustered in class a. The above suggests that not only did the genome polyploidization result in the increase of ANKTM gene number, but that tandem duplication is also a mechanism for the expansion of this subfamily. Micro-collinearity analysis of Triticeae ANKTMs indicates that some ANKTM type genes evolved into other types of ANKs in the evolution process. Public RNA-seq data showed that most of the genes in class d and class e are expressed, and some of them show differential responses to biotic stresses. Furthermore, qRT-PCR results showed that some ANKTMs in class d and class e responded to powdery mildew. Silencing of TaANKTM2A-5 by barley stripe mosaic virus-induced gene silencing compromised powdery mildew resistance in common wheat Bainongaikang58. Findings in this study not only help to understand the evolutionary process of ANKTM genes, but also form the basis for exploring disease resistance genes in the ANKTM gene family.

Proteomic and Phosphoproteomic Analyses Reveal a Complex Network Regulating Pollen Abortion and Potential Candidate Proteins in TCMS Wheat.

Thermosensitive sterile lines are natural materials for exploring the effects of anther development on male fertility. To study the possible molecular mechanisms regulating protein activity during the induction of male sterility, proteomic and phosphoproteomic analyses with tandem mass tags (TMTs) were used to study the binucleate anther of the thermosensitive sterile wheat line YS3038. A total of 9072 proteins, including 5019 phosphoproteins, were identified. Enrichment analyses of differentially abundant proteins (DAPs) and phosphoproteins (DAPPs) in metabolic pathways showed that both were mainly related to energy metabolism. Soluble sugar and ATP content were significantly decreased, free fatty acid content was significantly increased, and ROS was abnormally accumulated in male sterile YS3038-A. In addition, 233 kinase–substrate pairs involved in potential phosphorylation control networks were predicted to regulate fertility. Candidate proteins were identified, and a quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to validate the TMT results. TaPDCD5 is likely to be involved in fertility conversion of YS3038 by barley stripe mosaic virus-induced gene silencing (BSMV-VIGS). Our data provide new insights into the mechanism of TCMS, which has value for identifying potential candidate proteins associated with the formation or abortion of pollen and promotion of wheat heterosis utilization.

Genome-Wide Analysis of Serine Hydroxymethyltransferase Genes in Triticeae Species Reveals That TaSHMT3A-1 Regulates Fusarium Head Blight Resistance in Wheat.

Serine hydroxymethyltransferase (SHMT) plays a pivotal role in cellular one-carbon, photorespiration pathways and it influences the resistance to biotic and abiotic stresses. However, the function of SHMT proteins in wheat remains largely unexplored. In the present study, SHMT genes in five Triticeae species, Oryza sativa, and four dicotyledon species were identified based on whole genome information. The origin history of the target gene was traced by micro-collinearity analysis. Gene expression patterns of TaSHMTs in different tissues, various biotic stresses, exogenous hormones, and two biotic stresses were determined by Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The function of the selected TaSHMT3A-1 was studied by barley stripe mosaic virus-induced gene silencing in common wheat Bainong207. A total of 64 SHMT members were identified and further classified into two main classes based on the structure of SHMT proteins. The gene structure and motif composition analyses revealed that SHMTs kept relatively conserved within the same subclasses. Interestingly, there was a gene, TdSHMT7B-1, on chromosome 7B of Triticum dicoccoides, but there was no SHMT gene on chromosome 7 of other analyzed Triticeae species; TdSHMT7B-1 had fewer exons and conserved motifs than the genes in the same subclass, suggesting that the gene of TdSHMT7B-1 has a notable evolutionary progress. The micro-collinearity relationship showed that no homologs of TaSHMT3A-1 and its two neighboring genes were found in the collinearity region of Triticum urartu, and there were 27 genes inserted into the collinearity region of T. urartu. Furthermore, qRT-PCR results showed that TaSHMT3A-1 was responsive to abiotic stresses (NaCl and cold), abscisic acid, methyl jasmonate, and hydrogen peroxide. Significantly, upon Fusarium graminearum infection, the expression of TaSHMT3A-1 was highly upregulated in resistant cultivar Sumai3. More importantly, silencing of TaSHMT3A-1 compromises Fusarium head blight resistance in common wheat Bainong207. Our new findings suggest that the TaSHMT3A-1 gene in wheat plays an important role in resistance to Fusarium head blight. This provides a valuable reference for further study on the function of this gene family.

Chloroplast Genes Are Involved in The Male-Sterility of K-Type CMS in Wheat.

The utilization of crop heterosis can greatly improve crop yield. The sterile line is vital for the heterosis utilization of wheat (Triticum aestivum L.). The chloroplast genomes of two sterile lines and one maintainer were sequenced using second-generation high-throughput technology and assembled. The nonsynonymous mutated genes among the three varieties were identified, the expressed difference was further analyzed by qPCR, and finally, the function of the differentially expressed genes was analyzed by the barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) method. A total of 16 genes containing 31 nonsynonymous mutations between K519A and 519B were identified. There were no base mutations in the protein-encoding genes between K519A and YS3038. The chloroplast genomes of 519B and K519A were closely related to the Triticum genus and Aegilops genus, respectively. The gene expression levels of the six selected genes with nonsynonymous mutation sites for K519A compared to 519B were mostly downregulated at the binucleate and trinucleate stages of pollen development. The seed setting rates of atpB-silenced or ndhH-silenced 519B plants by BSMV-VIGS method were significantly reduced. It can be concluded that atpB and the ndhH are likely to be involved in the reproductive transformation of 519B.

A Ricin B-Like Lectin Protein Physically Interacts with TaPFT and Is Involved in Resistance to Fusarium Head Blight in Wheat.

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, has become one of the most serious diseases that damage wheat. The TaPFT (pore-forming toxin-like) and TaHRC (histidine-rich calcium-binding protein) genes at the quantitative trait locus Fhb1 were identified to confer resistance to FHB in the wheat cultivar Sumai 3. In this study, a wheat ricin B-like lectin gene (designated TaRBL) that interacted with TaPFT was isolated by a yeast two-hybrid screen of a wheat cDNA library. A yeast two-hybrid and bimolecular fluorescence complementation study further verified that TaRBL interacted with TaPFT but not with TaHRC. Gene expression studies showed that upon F. graminearum infection, TaRBL expression was upregulated in resistant cultivars but downregulated in susceptible cultivars. Furthermore, knockdown of TaRBL expression by barley stripe mosaic virus-induced gene silencing significantly reduced the resistance of wheat to FHB in both the resistant cultivar Sumai 3 and the susceptible cultivar Jimai 22. Thus, we conclude that TaRBL encodes a ricin B-like lectin protein that interacts with TaPFT and is involved in resistance to FHB in wheat.

Identification and verification of genes related to pollen development and male sterility induced by high temperature in the thermo-sensitive genic male sterile wheat line.

Bioinformatic analysis identified the function of genes regulating wheat fertility. Barley stripe mosaic virus-induced gene silencing verified that the genes TaMut11 and TaSF3 are involved in pollen development and related to fertility conversion. Environment-sensitive genic male sterility is of vital importance to hybrid vigor in crop production and breeding. Therefore, it is meaningful to study the function of the genes related to pollen development and male sterility, which is still not fully understand currently. In this study, YanZhan 4110S, a new thermo-sensitive genic male sterility wheat line, and its near-isogenic line YanZhan 4110 were analyzed. Through comparative transcriptome basic bioinformatics and weighted gene co-expression network to further identify some hub genes, the genes TaMut11 and TaSF3 associated with pollen development and male sterility induced by high-temperature were identified in YanZhan 4110S. Further verification through barley stripe mosaic virus-induced gene silencing elucidated that the silencing of TaMut11 and TaSF3 caused pollen abortion, finally resulting in the declination of fertility. These findings provided data on the abortive mechanism in environment-sensitive genic male sterility wheat.

Dynamic expression of miRNAs and functional analysis of target genes involved in the response to male sterility of the wheat line YS3038

Thermosensitive cytoplasmic male sterile (TCMS) lines play an important role in wheat breeding, heterosis utilization, and germplasm innovation. MicroRNAs (miRNAs) can regulate the expression level of target genes by inhibiting the translation of these genes. YS3038 is a wheat TCMS line. In this study, the fertility conversion mechanism of YS3038 was studied by examining the abortion characteristics of YS3038, the regulation pattern of miRNAs and the target genes of miRNAs in YS3038. MiRNA-seq was performed on three important stages of YS3038 under sterile and fertile conditions. Then, the clean reads were aligned with some databases to filter other ncRNAs and repeats. The known miRNAs and novel miRNAs were predicted by sequence comparison with known miRNAs from miRbase. Differential expression of miRNAs between different stages and between different fertile conditions was analyzed, and functional analysis of target genes with opposite expression patterns as those of the miRNAs was conducted. The Ubisch bodies and microspores of sterile anthers were covered with filamentous materials. The degradation of the tapetum cells, the chloroplast structure of endothecium cells, and the microspore structure were abnormal. Microspore development was hindered from the late uninucleate stage to the binucleate stage. Twenty, 52, and 68 differentially expressed miRNAs (DEmiRs) were identified at the early uninucleate, late uninucleate, and binucleate stages, respectively, and there were 0, 7, and 72 differentially expressed target genes (DETGs), respectively, at these three stages. At the binucleate stage, 29 DEmiRs had 41 target mRNAs in total, and the expression patterns of the 41 target mRNAs were opposite to those of the 29 miRNAs. Fifteen significantly enriched KEGG pathways were associated with the 41 target mRNAs. Leucine-rich repeat receptor-like kinases (LRR-RLKs) play important roles in plant developmental and physiological processes. Some studies have shown that the expression of LRR-RLKs is related to the differentiation of microsporocytes and tapetum cells and to male sterility. An LRR-RLK (TaeRPK) gene was silenced by the barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) method, and the seed setting rates of the TaeRPK-silenced plants (3.51%) were significantly lower than those of the negative control plants (88.78%) (P < 0.01). Thus, the TaeRPK gene is likely to be involved in the fertility conversion of YS3038.

In Silico Identification of the Full Complement of Subtilase-Encoding Genes and Characterization of the Role of TaSBT1.7 in Resistance Against Stripe Rust in Wheat.

Plant subtilases (SBTs) or subtilisin-like proteases comprise a very diverse family of serine peptidases that participates in a broad spectrum of biological functions. Despite increasing evidence for roles of SBTs in plant immunity in recent years, little is known about wheat (Triticum aestivum) SBTs (TaSBTs). Here, we identified 255 TaSBT genes from bread wheat using the latest version 2.0 of the reference genome sequence. The SBT family can be grouped into five clades, from TaSBT1 to TaSBT5, based on a phylogenetic tree constructed with deduced protein sequences. In silico protein-domain analysis revealed the existence of considerable sequence diversification of the TaSBT family which, together with the local clustered gene distribution, suggests that TaSBT genes have undergone extensive functional diversification. Among those TaSBT genes whose expression was altered by biotic factors, TaSBT1.7 was found to be induced in wheat leaves by chitin and flg22 elicitors, as well as six examined pathogens, implying a role for TaSBT1.7 in plant defense. Transient overexpression of TaSBT1.7 in Nicotiana benthamiana leaves resulted in necrotic cell death. Moreover, knocking down TaSBT1.7 in wheat using barley stripe mosaic virus-induced gene silencing compromised the hypersensitive response and resistance against Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust. Taken together, this study defined the full complement of wheat SBT genes and provided evidence for a positive role of one particular member, TaSBT1.7, in the incompatible interaction between wheat and a stripe rust pathogen.

Identification of ceRNA and candidate genes related to fertility conversion of TCMS line YS3038 in wheat

Previous studies have indicated that noncoding RNAs are important factors in gene functions. To explore the mechanism of male sterility of YS3038, the sterile genes were mapped, and based on previous work, the expression of long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), and their target genes was studied. Weighted gene coexpression network analysis (WGCNA) and competitive endogenous RNA (ceRNA) analysis were further performed for differentially expressed noncoding RNAs and target genes. At last, the candidate genes were silenced by barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) to prove their function. The sterile genes were mapped on chromosomes 1B and 6B based on chip mix pool analysis, and one major effect QTL (27.3190% variation) was found based on SSR primers. The WGCNA analysis revealed that the dark turquoise and steel blue modules were highly correlated with anther development and fertility conversion, respectively. The ceRNA analysis showed that a total of 184 RNAs interacted with each other, including 115 mRNAs, 55 microRNAs (miRNAs), eight circRNAs, and six lncRNAs. Finally, the seed setting rate of the plant was significantly decreased after fatty acyl-CoA reductase 5 silencing. This study provides breeders with a new option for the development of thermosensitive cytoplasmic male-sterile (TCMS) wheat lines, which will favor the sustainable development of two-line hybrid wheat.

Silencing of the receptor-like cytoplasmic kinase gene TaRKL1 reduces photosynthetic capacity in wheat

To explore the role of receptor-like kinases in the regulation of photosynthesis, an uncharacterized TaRKL1 encoding a receptor-like cytoplasmic kinase was investigated in Triticum aestivum cv. Xiaoyan 101 with Barley stripe mosaic virus-induced gene silencing system (BSMV-VIGS). The results showed that the CO2 assimilation rate, stomatal conductance, and transpiration rate were significantly lower in the BSMV:TaRKL1 plants than those in the BSMV:γ00 plants (control). Moreover, the maximum photochemical efficiency and electron transport flux decreased while the dissipated energy flux was enhanced in the BSMV:TaRKL1 plants compared to the control. Additionally, the contents of chlorophylls and carotenoids were reduced in the BSMV:TaRKL1 plants. However, the hydrogen peroxide content was significantly enhanced in the BSMV:TaRKL1 plants, which resulted from lower ascorbate peroxidase activity. Consistent with the inhibition of photosynthesis, the transcription levels of the photosynthesis-related, antioxidant enzymes, senescence-associated genes, and four abscisic acid biosynthesis genes were downregulated substantially. Collectively, this study for the first time showed that TaRKL1 regulates photosynthesis and H2O2 homeostasis. It may be a potential target gene for radiation-use efficiency improvement in wheat.

Overexpression of HvAKT1 improves drought tolerance in barley by regulating root ion homeostasis and ROS and NO signaling.

Potassium (K+) is the major cationic inorganic nutrient utilized for osmotic regulation, cell growth, and enzyme activation in plants. Inwardly rectifying K+ channel 1 (AKT1) is the primary channel for root K+ uptake in plants, but the function of HvAKT1 in barley plants under drought stress has not been fully elucidated. In this study, we conducted evolutionary bioinformatics, biotechnological, electrophysiological, and biochemical assays to explore molecular mechanisms of HvAKT1 in response to drought in barley. The expression of HvAKT1 was significantly up-regulated by drought stress in the roots of XZ5-a drought-tolerant wild barley genotype. We isolated and functionally characterized the plasma membrane-localized HvAKT1 using Agrobacterium-mediated plant transformation and Barley stripe mosaic virus-induced gene silencing of HvAKT1 in barley. Evolutionary bioinformatics indicated that the K+ selective filter in AKT1 originated from streptophyte algae and is evolutionarily conserved in land plants. Silencing of HvAKT1 resulted in significantly decreased biomass and suppressed K+ uptake in root epidermal cells under drought treatment. Disruption of HvAKT1 decreased root H+ efflux, H+-ATPase activity, and nitric oxide (NO) synthesis, but increased hydrogen peroxide (H2O2) production in the roots under drought stress. Furthermore, we observed that overexpression of HvAKT1 improves K+ uptake and increases drought resistance in barley. Our results highlight the importance of HvAKT1 for root K+ uptake and its pleiotropic effects on root H+-ATPase, and H2O2 and NO in response to drought stress, providing new insights into the genetic basis of drought tolerance and K+ nutrition in barley.

HvHOX9, a novel homeobox leucine zipper transcription factor, positively regulates aluminum tolerance in Tibetan wild barley.

Aluminum (Al) toxicity is the primary limiting factor of crop production on acid soils. Tibetan wild barley germplasm is a valuable source of potential genes for breeding barley with acid and Al tolerance. We performed microRNA and RNA sequencing using wild (XZ16, Al-tolerant; XZ61, Al-sensitive) and cultivated (Dayton, Al-tolerant) barley. A novel homeobox-leucine zipper transcription factor, HvHOX9, was identified as a target gene of miR166b and functionally characterized. HvHOX9 was up-regulated by Al stress in XZ16 (but unchanged in XZ61 and Dayton) and was significantly induced only in root tip. Phylogenetic analysis showed that HvHOX9 is most closely related to wheat TaHOX9 and orthologues of HvHOX9 are present in the closest algal relatives of Zygnematophyceae. Barley stripe mosaic virus-induced gene silencing of HvHOX9 in XZ16 led to significantly increased Al sensitivity but did not affect its sensitivity to other metals and low pH. Disruption of HvHOX9 did not change Al concentration in the root cell sap, but led to more Al accumulation in root cell wall after Al exposure. Silencing of HvHOX9 decreased H+ influx after Al exposure. Our findings suggest that miR166b/HvHOX9 play a critical role in Al tolerance by decreasing root cell wall Al binding and increasing apoplastic pH for Al detoxification in the root.