Identification of nuclear membrane SUN proteins and components associated with wheat fungal stress responses.

In eukaryotes, the nuclear membrane that encapsulates genomic DNA is composed of an inner nuclear membrane (INM), an outer nuclear membrane (ONM), and a perinuclear space. SUN proteins located in the INM and KASH proteins in the ONM form the SUN-KASH NM-bridge, which functions as the junction of the nucleocytoplasmic complex junction. Proteins containing the SUN domain showed the highest correlation with differentially accumulated proteins (DAPs) in the wheat response to fungal stress. To understand the characteristics of SUN and its associated proteins in wheat responding to pathogen stress, here we investigated and comprehensive analyzed SUN- and KASH-related proteins among the DAPs under fungi infection based on their conserved motifs. In total, four SUN proteins, one WPP domain-interacting protein (WIP), four WPP domain-interacting tail-anchored proteins (WIT), two WPP proteins and one Ran GTPase activating protein (RanGAP) were identified. Following transient expression of Nicotiana benthamiana, TaSUN2, TaRanGAP2, TaWIT1 and TaWIP1 were identified as nuclear membrane proteins, while TaWPP1 and TaWPP2 were expressed in both the nucleus and cell membrane. RT-qPCR analysis demonstrated that the transcription of TaSUN2, TaRanGAP2 and TaWPP1 were strongly upregulated in response to fungal infection. Furthermore, using the bimolecular fluorescence complementation, the luciferase complementation and a nuclear and split-ubiquitin-based membrane yeast two-hybrid systems, we substantiated the interaction between TaSUN2 and TaWIP1, as well as TaWIP1/WIT1 and TaWPP1/WPP2. Silencing of TaSUN2, TaRanGAP2 and TaWPP1 in wheat leaves promoted powdery mildew infection and hyphal growth, and reduced the expression of TaBRI1, TaBAK1 and Ta14-3-3, indicating that these NM proteins play a positive role in resistance to fungal stress. Our study reveals the characteristics of NM proteins and propose the preliminary construction of SUN-WIP-WPP-RanGAP complex in wheat, which represents a foundation for detail elucidating their functions in wheat in future.

Population-level transcriptomes reveal gene expression and splicing underlying cadmium accumulation in barley.

Genetic variation is an important determinant of gene transcription, which in turn contributes to functional and phenotypic diversity. Identification of the genetic variants controlling gene expression and alternative splicing in crops responding to cadmium (Cd), an important issue for food safety and human health, is of great value to improve our understanding of Cd accumulation-related genes. Here we report an in-depth survey of population-level transcriptome variation of barley (Hordeum vulgare) core accessions under Cd exposure. We reveal marked transcriptomic changes in response to Cd exposure, and these are largely independent of tissues. A genome-wide association study (GWAS) revealed 59 498 expression quantitative trait loci (eQTLs) and 23 854 splicing quantitative trait loci (sQTLs), leading to a complex network that covers 66.6% of the expressed genes, including 68 metal transporter genes. On average, 41.0% of sQTLs overlapped with eQTLs across different tissues, indicating that these two dimensions of transcript variation are largely independent. Moreover, we found that 34.5% of GWAS QTLs that underlie 10 Cd accumulation traits in barley are co-localized with eQTLs and sQTLs, which could imply a mechanistic role of different genetic variants affecting gene expression and alternative splicing in these traits. This study highlights the role of distal and proximal genetic effects on gene expression, splicing, and phenotypic plasticity. We anticipate that our results on the genetic control of expression and splicing underlying Cd accumulation provide a bridge to better understand genetic variation and phenotypic diversity to elucidate the mechanisms underlying Cd accumulation in plants.

An alternative splicing isoform of wheat TaYRG1 resistance protein activates immunity by interacting with dynamin-related proteins.

Wheat (Triticum aestivum) is a commercially important crop and its production is seriously threatened by the fungal pathogen Puccinia striiformis f. sp. tritici West (Pst). Resistance (R) genes are critical factors that facilitate plant immune responses. Here, we report a wheat R gene NB-ARC-LRR ortholog, TaYRG1, that is associated with distinct alternative splicing events in wheat infected by Pst. The native splice variant, TaYRG1.6, encodes internal-motif-deleted polypeptides with the same N- and C-termini as TaYRG1.1, resulting in gain of function. Transient expression of protein variants in Nicotiana benthamiana showed that the NB and ARC domains, and TaYRG1.6 (half LRR domain), stimulate robust elicitor-independent cell death based on a signal peptide, although the activity was negatively modulated by the CC and complete LRR domains. Furthermore, molecular genetic analyses indicated that TaYRG1.6 enhanced resistance to Pst in wheat. Moreover, we provide multiple lines of evidence that TaYRG1.6 interacts with a dynamin-related protein, TaDrp1. Proteome profiling suggested that the TaYRG1.6-TaDrp1-DNM complex in the membrane trafficking systems may trigger cell death by mobilizing lipid and kinase signaling in the endocytosis pathway. Our findings reveal a unique mechanism by which TaYRG1 activates cell death and enhances disease resistance by reconfiguring protein structure through alternative splicing.

Characteristics and Expression Analysis of Invertase Gene Family in Common Wheat (Triticum aestivum L.).

Invertase (INV) irreversibly catalyzes the conversion of sucrose into glucose and fructose, playing important role in plant development and stress tolerance. However, the functions of INV genes in wheat have been less studied. In this study, a total of 126 TaINV genes were identified using a genome-wide search method, which could be classified into five classes (TaCWI-α, TaCWI-ß, TaCI-α, TaCI-ß, and TaVI) based on phylogenetic relationship. A total of 101 TaINVs were collinear with their ancestors in the synteny analysis, and we speculated that polyploidy events were the main force in the expansion of the TaINV gene family. Compared with TaCI, TaCWI and TaVI are more similar in gene structure and protein properties. Transcriptome sequencing analysis showed that TaINVs expressed in multiple tissues with different expression levels. Among 19 tissue-specific expressed TaINVs, 12 TaINVs showed grain-specific expression pattern and might play an important role in wheat grain development. In addition, qRT-PCR results further confirmed that TaCWI50 and TaVI27 show different expression in grain weight NILs. Our results demonstrated that the high expression of TaCWI50 and TaVI27 may be associated with a larger TGW phenotype. This work provides the foundations for understanding the grain development mechanism.

Introgression of the Aegilops speltoides Su1-Ph1 Suppressor into Wheat.

Meiotic pairing between homoeologous chromosomes in polyploid wheat is inhibited by the Ph1 locus on the long arm of chromosome 5 in the B genome. Aegilops speltoides (genomes SS), the closest relative of the progenitor of the wheat B genome, is polymorphic for genetic suppression of Ph1. Using this polymorphism, two major suppressor loci, Su1-Ph1 and Su2-Ph1, have been mapped in Ae. speltoides. Su1-Ph1 is located in the distal, high-recombination region of the long arm of the Ae. speltoides chromosome 3S. Its location and tight linkage to marker Xpsr1205-3S makes Su1-Ph1 a suitable target for introgression into wheat. Here, Xpsr1205-3S was introgressed into hexaploid bread wheat cv. Chinese Spring (CS) and from there into tetraploid durum wheat cv. Langdon (LDN). Sequential fluorescence in situ hybridization and genomic in situ hybridization showed that an Ae. speltoides segment with Xpsr1205-3S replaced the distal end of the long arm of chromosome 3A. In the CS genetic background, the chromosome induced homoeologous chromosome pairing in interspecific hybrids with Ae. peregrina but not in progenies from crosses involving alien disomic substitution lines. In the LDN genetic background, the chromosome induced homoeologous chromosome pairing in both interspecific hybrids and progenies from crosses involving alien disomic substitution lines. We conclude that the recombined chromosome harbors Su1-Ph1 but its expression requires expression of complementary gene that is present in LDN but absent in CS. We suggest that it is unlikely that Su1-Ph1 and ZIP4-1, a paralog of Ph1 located on wheat chromosomes 3A and 3B and Ae. tauschii chromosome 3D, are equivalent. The utility of Su1-Ph1 for induction of recombination between homoeologous chromosomes in wheat is illustrated.

Chromosomal structural changes and microsatellite variations in newly synthesized hexaploid wheat mediated by unreduced gametes.

Allohexaploid wheat was derived from interspecific hybridization, followed by spontaneous chromosome doubling. Newly synthesized hexaploid wheat by crossing Triticum turgidum and Aegilops tauschii provides a classical model to understand the mechanisms of allohexaploidization in wheat. However, immediate chromosome level variation and microsatellite level variation of newly synthesized hexaploid wheat have been rarely reported. Here, unreduced gametes were applied to develop synthesized hexaploid wheat, NA0928, population by crossing T. turgidum ssp. dicoccum MY3478 and Ae. tauschii SY41, and further S0-S3 generations of NA0928 were assayed by sequential cytological and microsatellite techniques. We demonstrated that plentiful chromosomal structural changes and microsatellite variations emerged in the early generations of newly synthesized hexaploid wheat population NA0928, including aneuploidy with whole-chromosome loss or gain, aneuploidy with telosome formation, chromosome-specific repeated sequence elimination (indicated by fluorescence in situ hybridization) and microsatellite sequence elimination (indicated by sequencing), and many kinds of variations have not been previously reported. Additionally, we reported a new germplasm, T. turgidum accession MY3478 with excellent unreduced gametes trait, and then succeeded to transfer powdery mildew resistance from Ae. tauschii SY41 to synthesized allohexaploid wheat population NA0928, which would be valuable resistance resources for wheat improvement.

Quantitative proteomics reveals the central changes of wheat in response to powdery mildew.

UNLABELLED: Powdery mildew (Pm), caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most important crop diseases, causing severe economic losses to wheat production worldwide. However, there are few reports about the proteomic response to Bgt infection in resistant wheat. Hence, quantitative proteomic analysis of N9134, a resistant wheat line, was performed to explore the molecular mechanism of wheat in defense against Bgt. Comparing the leaf proteins of Bgt-inoculated N9134 with that of mock-inoculated controls, a total of 2182 protein-species were quantified by iTRAQ at 24, 48 and 72h postinoculation (hpi) with Bgt, of which 394 showed differential accumulation. These differentially accumulated protein-species (DAPs) mainly included pathogenesis-related (PR) polypeptides, oxidative stress responsive proteins and components involved in primary metabolic pathways. KEGG enrichment analysis showed that phenylpropanoid biosynthesis, phenylalanine metabolism and photosynthesis-antenna proteins were the key pathways in response to Bgt infection. InterProScan 5 and the Gibbs Motif Sampler cluster 394 DAPs into eight conserved motifs, which shared leucine repeats and histidine sites in the sequence motifs. Moreover, eight separate protein-protein interaction (PPI) networks were predicted from STRING database. This study provides a powerful platform for further exploration of the molecular mechanism underlying resistant wheat responding to Bgt. SIGNIFICANCE: Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive pathogenic disease in wheat-producing regions worldwide, resulting in severe yield reductions. Although many resistant wheat varieties have been cultivated, there are few reports about the proteomic response to Bgt infection in resistant wheat. Therefore, an iTRAQ-based quantitative proteomic analysis of a resistant wheat line (N9134) in response to Bgt infection has been performed. This paper provides new insights into the underlying molecular mechanism of wheat in response to Bgt. The proteomic analysis can significantly narrow the field of potential defense-related protein-species, and is conducive to recognize the critical or effector protein under Bgt infection more precisely. Taken together, large amounts of high-throughput data provide a powerful platform for further exploration of the molecular mechanism on wheat-Bgt interactions.

Spontaneous and divergent hexaploid triticales derived from common wheat × rye by complete elimination of D-genome chromosomes.

BACKGROUND: Hexaploid triticale could be either synthesized by crossing tetraploid wheat with rye, or developed by crossing hexaploid wheat with a hexaploid triticale or an octoploid triticale. METHODOLOGY/PRINCIPAL FINDINGS: Here two hexaploid triticales with great morphologic divergence derived from common wheat cultivar M8003 (Triticum aestivum L.) × Austrian rye (Secale cereale L.) were reported, exhibiting high resistance for powdery mildew and stripe rust and potential for wheat improvement. Sequential fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) karyotyping revealed that D-genome chromosomes were completely eliminated and the whole A-genome, B-genome and R-genome chromosomes were retained in both lines. Furthermore, plentiful alterations of wheat chromosomes including 5A and 7B were detected in both triticales and additionally altered 5B, 7A chromosome and restructured chromosome 2A was assayed in N9116H and N9116M, respectively, even after selfing for several decades. Besides, meiotic asynchrony was displayed and a variety of storage protein variations were assayed, especially in the HMW/LMW-GS region and secalins region in both triticales. CONCLUSION: This study confirms that whole D-genome chromosomes could be preferentially eliminated in the hybrid of common wheat × rye, "genome shock" was accompanying the allopolyploidization of nascent triticales, and great morphologic divergence might result from the genetic variations. Moreover, new hexaploid triticale lines contributing potential resistance resources for wheat improvement were produced.

Isolation and characterization of a wheat IF2 homolog required for innate immunity to stripe rust.

KEY MESSAGE: The wheat eIF2 homolog, TaIF2, is induced by the stripe rust pathogen CYR 32 at an early stage of inoculation and is related to the innate immunity resistance level in wheat. The initiation of translation represents a critical control point in the regulation of gene expression in all organisms. We previously identified an upregulated EST S186 (EL773056) from an SSH-cDNA library of the Shaanmai 139 strain of wheat (Triticum aestivum) infected with Puccinia striiformis (Pst). In the present work, we isolated a cDNA clone and identified it as a wheat IF2 homolog. This cDNA consisted of 1,314 nucleotides and contained an open reading frame of 795 nucleotides encoding a polypeptide of 254 amino acids. The amino acids represent a conserved domain in EF-Tu, mtIF2-II, and mtIF2-Ivc. The alignment result showed that it maybe a partial cDNA of the initiation factor 2/eukaryotic initiation factor 5B (IF2/eIF5B) superfamily gene. Paradoxically, results of a Swiss-model analysis suggesting a low QMEAN Z-score implied that it was a membrane protein. Quantitative RT-PCR studies confirmed that the wheat eIF2 (TaIF2) homolog was differentially expressed in three near-isogenic lines. Critical time points for the induction of resistance by inoculation with Pst CYR32 in YrSM139-1B + YrSM139-2D immune resistance genotype occurred at 1 and 3 dpi (days post-infection). RNAi test showed that the inoculated BSMV-IF2 leaves of Shaanmai 139 showed obvious cell death after 15 days of inoculation with CYR 32. qRT-PCR analysis of the target gene in cDNA samples isolated from BSMV-IF2-Pst, BSMV-0-Pst and Pst infected leaves confirmed that the expression of TaIF2 is suppressed by BSMV-IF2 at 3 dpi. This suggested that TaIF2/eIF5B plays an important role in the mechanism of innate immunity to stripe rust pathogen.