Transfection of entomopathogenic Metarhizium species with a mycovirus confers hypervirulence against two lepidopteran pests.

Although most known viruses infecting fungi pathogenic to higher eukaryotes are asymptomatic or reduce the virulence of their host fungi, those that confer hypervirulence to entomopathogenic fungus still need to be explored. Here, we identified and studied a novel mycovirus in Metarhizium flavoviride, isolated from small brown planthopper (Laodelphax striatellus). Based on molecular analysis, we tentatively designated the mycovirus as Metarhizium flavoviride partitivirus 1 (MfPV1), a species in genus Gammapartitivirus, family Partitiviridae. MfPV1 has two double-stranded RNAs as its genome, 1,775 and 1,575 bp in size respectively, encapsidated in isometric particles. When we transfected commercial strains of Metarhizium anisopliae and Metarhizium pingshaense with MfPV1, conidiation was significantly enhanced (t test; P-value < 0. 01), and the significantly higher mortality rates of the larvae of diamondback moth (Plutella xylostella) and fall armyworm (Spodoptera frugiperda), two important lepidopteran pests were found in virus-transfected strains (ANOVA; P-value < 0.05). Transcriptomic analysis showed that transcript levels of pathogenesis-related genes in MfPV1-infected M. anisopliae were obviously altered, suggesting increased production of metarhizium adhesin-like protein, hydrolyzed protein, and destruxin synthetase. Further studies are required to elucidate the mechanism whereby MfPV1 enhances the expression of pathogenesis-related genes and virulence of Metarhizium to lepidopteran pests. This study presents experimental evidence that the transfection of other entomopathogenic fungal species with a mycovirus can confer significant hypervirulence and provides a good example that mycoviruses could be used as a synergistic agent to enhance the biocontrol activity of entomopathogenic fungi.

Barley GRIK1-SnRK1 kinases subvert a viral virulence protein to upregulate antiviral RNAi and inhibit infection.

Viruses often usurp host machineries for their amplification, but it remains unclear if hosts may subvert virus proteins to regulate viral proliferation. Here, we show that the 17K protein, an important virulence factor conserved in barley yellow dwarf viruses (BYDVs) and related poleroviruses, is phosphorylated by host GRIK1-SnRK1 kinases, with the phosphorylated 17K (P17K) capable of enhancing the abundance of virus-derived small interfering RNAs (vsiRNAs) and thus antiviral RNAi. Furthermore, P17K interacts with barley small RNA-degrading nuclease 1 (HvSDN1) and impedes HvSDN1-catalyzed vsiRNA degradation. Additionally, P17K weakens the HvSDN1-HvAGO1 interaction, thus hindering HvSDN1 from accessing and degrading HvAGO1-carried vsiRNAs. Importantly, transgenic expression of 17K phosphomimetics (17K5D ), or genome editing of SDN1, generates stable resistance to BYDV through elevating vsiRNA abundance. These data validate a novel mechanism that enhances antiviral RNAi through host subversion of a viral virulence protein to inhibit SDN1-catalyzed vsiRNA degradation and suggest new ways for engineering BYDV-resistant crops.

Appropriate reduction of importin-α gene expression enhances yellow dwarf disease resistance in common wheat.

Barley yellow dwarf viruses (BYDVs) cause widespread damage to global cereal crops. Here we report a novel strategy for elevating resistance to BYDV infection. The 17K protein, a potent virulence factor conserved in BYDVs, interacted with barley IMP-α1 and -α2 proteins that are nuclear transport receptors. Consistently, a nuclear localization signal was predicted in 17K, which was found essential for 17K to be transported into the nucleus and to interact with IMP-α1 and -α2. Reducing HvIMP-α1 and -α2 expression by gene silencing attenuated BYDV-elicited dwarfism, accompanied by a lowered nuclear accumulation of 17K. Among the eight common wheat CRISPR mutants with two to four TaIMP-α1 and -α2 genes mutated, the triple mutant α1aaBBDD /α2AAbbdd and the tetra-mutant α1aabbdd /α2AAbbDD displayed strong BYDV resistance without negative effects on plant growth under field conditions. The BYDV resistance exhibited by α1aaBBDD /α2AAbbdd and α1aabbdd /α2AAbbDD was correlated with decreased nuclear accumulation of 17K and lowered viral proliferation in infected plants. Our work uncovers the function of host IMP-α proteins in BYDV pathogenesis and generates the germplasm valuable for breeding BYDV-resistant wheat. Appropriate reduction of IMP-α gene expression may be broadly useful for enhancing antiviral resistance in agricultural crops and other economically important organisms.

Genomic and biological characteristics of a novel leafhopper-transmitted marafivirus infecting Triticum aestivum.

Here, we report a novel wheat-infecting marafivirus, tentatively named "Triticum aestivum marafivirus" (TaMRV). The full-length genome sequence of TaMRV comprises 6,437 nucleotides, excluding the poly(A) tail. Pairwise sequence comparisons and phylogenetic analysis revealed that TaMRV may represent a novel species within the genus Marafivirus in the family Tymoviridae. We also observed a mass of isometric particles with a diameter of about 30 nm in ultrathin sections of infected wheat leaf tissue. In addition, the leafhopper Psammotettix alienus was identified as a vector for this virus. This is the first report of the occurrence of a wheat-infecting marafivirus.

Efficient expression and function of a receptor-like kinase in wheat powdery mildew defence require an intron-located MYB binding site.

The LRK10-like receptor kinases (LRK10L-RLKs) are ubiquitously present in higher plants, but knowledge of their expression and function is still limited. Here, we report expression and functional analysis of TtdLRK10L-1, a typical LRK10L-RLK in durum wheat (Triticum turgidum L. ssp. durum). The introns of TtdLRK10L-1 contained multiple kinds of predicted cis-elements. To investigate the potential effect of these cis-elements on TtdLRK10L-1 expression and function, two types of transgenic wheat lines were prepared, which expressed a GFP-tagged TtdLRK10L-1 protein (TtdLRK10L-1:GFP) from the cDNA or genomic DNA (gDNA) sequence of TtdLRK10L-1 under the native promoter. TtdLRK10L-1:GFP expression was up-regulated by the powdery mildew pathogen Blumeria graminis f. sp. tritici (Bgt) in both types of transgenic plants, with the scale of the elevation being much stronger in the gDNA lines. Both types of transgenic plants exhibited enhanced resistance to Bgt infection relative to wild type control. Notably, the Bgt defence activated in the gDNA lines was significantly stronger than that in the cDNA lines. Further analysis revealed that a putative MYB transcription factor binding site (MYB-BS, CAGTTA) located in TtdLRK10L-1 intron I was critical for the efficient expression and function of TtdLRK10L-1 in Bgt defence. This MYB-BS could also increase the activity of a superpromoter widely used in ectopic gene expression studies in plants. Together, our results deepen the understanding of the expression and functional characteristics of LRK10L-RLKs. TtdLRK10L-1 is likely useful for further dissecting the molecular processes underlying wheat defence against Bgt and for developing Bgt resistant wheat crops.

Wheat heat tolerance is impaired by heightened deletions in the distal end of 4AL chromosomal arm.

Heat stress (HS) causes substantial damages to worldwide crop production. As a cool season crop, wheat (Triticum aestivum) is sensitive to HS-induced damages. To support the genetic improvement of wheat HS tolerance (HST), we conducted fine mapping of TaHST1, a locus required for maintaining wheat vegetative and reproductive growth under elevated temperatures. TaHST1 was mapped to the distal terminus of 4AL chromosome arm using genetic populations derived from two BC6 F6 breeding lines showing tolerance (E6015-4T) or sensitivity (E6015-3S) to HS. The 4AL region carrying TaHST1 locus was approximately 0.949 Mbp and contained the last 19 high confidence genes of 4AL according to wheat reference genome sequence. Resequencing of E6015-3S and E6015-4T and haplotype analysis of 3087 worldwide wheat accessions revealed heightened deletion polymorphisms in the distal 0.949 Mbp region of 4AL, which was confirmed by the finding of frequent gene losses in this region in eight genome-sequenced hexaploid wheat cultivars. The great majority (86.36%) of the 3087 lines displayed different degrees of nucleotide sequence deletions, with only 13.64% of them resembling E6015-4T in this region. These deletions can impair the presence and/or function of TaHST1 and surrounding genes, thus rendering global wheat germplasm vulnerable to HS or other environmental adversities. Therefore, conscientious and urgent efforts are needed in global wheat breeding programmes to optimize the structure and function of 4AL distal terminus by ensuring the presence of TaHST1 and surrounding genes. The new information reported here will help to accelerate the ongoing global efforts in improving wheat HST.

The TuMYB46L-TuACO3 module regulates ethylene biosynthesis in einkorn wheat defense to powdery mildew.

Powdery mildew disease, elicited by the obligate fungal pathogen Blumeria graminis f.sp. tritici (Bgt), causes widespread yield losses in global wheat crop. However, the molecular mechanisms governing wheat defense to Bgt are still not well understood. Here we found that TuACO3, encoding the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase functioning in ethylene (ET) biosynthesis, was induced by Bgt infection of the einkorn wheat Triticum urartu, which was accompanied by increased ET content. Silencing TuACO3 decreased ET production and compromised wheat defense to Bgt, whereas both processes were enhanced in the transgenic wheat overexpressing TuACO3. TuMYB46L, phylogenetically related to Arabidopsis MYB transcription factor AtMYB46, was found to bind to the TuACO3 promoter region in yeast-one-hybrid and EMSA experiments. TuMYB46L expression decreased rapidly following Bgt infection. Silencing TuMYB46L promoted ET content and Bgt defense, but the reverse was observed when TuMYB46L was overexpressed. Hence, decreased expression of TuMYB46L permits elevated function of TuACO3 in ET biosynthesis in Bgt-infected wheat. The TuMYB46L-TuACO3 module regulates ET biosynthesis to promote einkorn wheat defense against Bgt. Furthermore, we found four chitinase genes acting downstream of the TuMYB46L-TuACO3 module. Collectively, our data shed a new light on the molecular mechanisms underlying wheat defense to Bgt.

Analysis of the Gli-D2 locus identifies a genetic target for simultaneously improving the breadmaking and health-related traits of common wheat.

Gliadins are a major component of wheat seed proteins. However, the complex homoeologous Gli-2 loci (Gli-A2, -B2 and -D2) that encode the α-gliadins in commercial wheat are still poorly understood. Here we analyzed the Gli-D2 locus of Xiaoyan 81 (Xy81), a winter wheat cultivar. A total of 421.091 kb of the Gli-D2 sequence was assembled from sequencing multiple bacterial artificial clones, and 10 α-gliadin genes were annotated. Comparative genomic analysis showed that Xy81 carried only eight of the α-gliadin genes of the D genome donor Aegilops tauschii, with two of them each experiencing a tandem duplication. A mutant line lacking Gli-D2 (DLGliD2) consistently exhibited better breadmaking quality and dough functionalities than its progenitor Xy81, but without penalties in other agronomic traits. It also had an elevated lysine content in the grains. Transcriptome analysis verified the lack of Gli-D2 α-gliadin gene expression in DLGliD2. Furthermore, the transcript and protein levels of protein disulfide isomerase were both upregulated in DLGliD2 grains. Consistent with this finding, DLGliD2 had increased disulfide content in the flour. Our work sheds light on the structure and function of Gli-D2 in commercial wheat, and suggests that the removal of Gli-D2 and the gliadins specified by it is likely to be useful for simultaneously enhancing the end-use and health-related traits of common wheat. Because gliadins and homologous proteins are widely present in grass species, the strategy and information reported here may be broadly useful for improving the quality traits of diverse cereal crops.

Deletion of a target gene in Indica rice via CRISPR/Cas9.

KEY MESSAGE: Using CRISPR/Cas9, we successfully deleted large fragments of the yield-related gene DENSE AND ERECT PANICLE1 in Indica rice at relatively high frequency and generated gain-of-function dep1 mutants. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is a rapidly developing technology used to produce gene-specific modifications in both mammalian and plant systems. Most CRISPR-induced modifications in plants reported to date have been small insertions or deletions. Few large target gene deletions have thus far been reported, especially for Indica rice. In this study, we designed multiple CRISPR sgRNAs and successfully deleted DNA fragments in the gene DENSE AND ERECT PANICLE1 (DEP1) in the elite Indica rice line IR58025B. We achieved deletion frequencies of up to 21% for a 430 bp target and 9% for a 10 kb target among T0 events. Constructs with four sgRNAs did not generate higher full-length deletion frequencies than constructs with two sgRNAs. The multiple mutagenesis frequency reached 93% for four targets, and the homozygous mutation frequency reached 21% at the T0 stage. Important yield-related trait characteristics, such as dense and erect panicles and reduced plant height, were observed in dep1 homozygous T0 mutant plants produced by CRISPR/Cas9. Therefore, we successfully obtained deletions in DEP1 in the Indica background using the CRISPR/Cas9 editing tool at relatively high frequency.

N6-methyladenosine modification of the mRNA for a key gene in purine nucleotide metabolism regulates virus proliferation in an insect vector.

The titer of viruses that persist and propagate in their insect vector must be high enough for transmission yet not harm the insect, but the mechanism of this dynamic balance is unclear. Here, expression of inosine monophosphate dehydrogenase (LsIMPDH), a rate-limiting enzyme for guanosine triphosphate (GTP) synthesis, is shown to be downregulated by increased levels of N6-methyladenosine (m6A) on LsIMPDH mRNA in rice stripe virus (RSV)-infected small brown planthoppers (SBPHs; Laodelphax striatellus), the RSV vector, which decreases GTP content, thus limiting viral proliferation. Moreover, planthopper methyltransferase-like protein 3 (LsMETTL3) and m6A reader protein LsYTHDF3 are found to catalyze and recognize the m6A on LsIMPDH mRNA, respectively, and cooperate in destabilizing LsIMPDH transcripts. Co-silencing assays show that negative regulation of viral proliferation by both LsMETTL3 and LsYTHDF3 is partially dependent on LsIMPDH. This distinct mechanism limits virus replication in an insect vector, providing a potential gene target to block viral transmission.

CRISPR/Cas9-Mediated Resistance to Wheat Dwarf Virus in Hexaploid Wheat (Triticum aestivum L.).

Wheat dwarf virus (WDV, genus Mastrevirus, family Geminiviridae) is one of the causal agents of wheat viral disease, which severely impacts wheat production in most wheat-growing regions in the world. Currently, there is little information about natural resistance against WDV in common wheat germplasms. CRISPR/Cas9 technology is being utilized to manufacture transgenic plants resistant to different diseases. In the present study, we used the CRISPR/Cas9 system targeting overlapping regions of coat protein (CP) and movement protein (MP) (referred to as CP/MP) or large intergenic region (LIR) in the wheat variety 'Fielder' to develop resistance against WDV. WDV-inoculated T1 progenies expressing Cas9 and sgRNA for CP/MP and LIR showed complete resistance against WDV and no accumulation of viral DNA compared with control plants. Mutation analysis revealed that the CP/MP and LIR targeting sites have small indels in the corresponding Cas9-positive plants. Additionally, virus inhibition and indel mutations occurred in T2 homozygous lines. Together, our work gives efficient results of the engineering of CRISPR/Cas9-mediated WDV resistance in common wheat plants, and the specific sgRNAs identified in this study can be extended to utilize the CRISPR/Cas9 system to confer resistance to WDV in other cereal crops such as barley, oats, and rye.

A novel module regulating ROS in NLR-mediated immunity.

The regulatory mechanisms of apoplastic reactive oxygen species (ROS) production during pattern-triggered immunity (PTI) are well known. However, how ROS levels are regulated during effector-triggered immunity (ETI) remains largely unknown. Recently, Zhang et al. discovered that MAPK-Alfin-like 7 module enhances nucleotide-binding, leucine-rich repeat receptor (NLR)-mediated immunity by negatively regulating genes encoding ROS scavenging enzymes, deepening our understanding of ROS control during ETI in plants.

CRISPR/LbCas12a-Mediated Genome Editing in Soybean.

Currently methods for generating soybean edited lines are time-consuming, inefficient, and limited to certain genotypes. Here we describe a fast and highly efficient genome editing method based on CRISPR-Cas12a nuclease system in soybean. The method uses Agrobacterium-mediated transformation to deliver editing constructs and uses aadA or ALS genes as selectable marker. It only takes about 45 days to obtain greenhouse-ready edited plants at higher than 30% transformation efficiency and 50% editing rate. The method is applicable to other selectable markers including EPSPS and has low transgene chimera rate. The method is also genotype-flexible and has been applied to genome editing of several elite soybean varieties.

A high-quality genome assembly highlights rye genomic characteristics and agronomically important genes.

Rye is a valuable food and forage crop, an important genetic resource for wheat and triticale improvement and an indispensable material for efficient comparative genomic studies in grasses. Here, we sequenced the genome of Weining rye, an elite Chinese rye variety. The assembled contigs (7.74 Gb) accounted for 98.47% of the estimated genome size (7.86 Gb), with 93.67% of the contigs (7.25 Gb) assigned to seven chromosomes. Repetitive elements constituted 90.31% of the assembled genome. Compared to previously sequenced Triticeae genomes, Daniela, Sumaya and Sumana retrotransposons showed strong expansion in rye. Further analyses of the Weining assembly shed new light on genome-wide gene duplications and their impact on starch biosynthesis genes, physical organization of complex prolamin loci, gene expression features underlying early heading trait and putative domestication-associated chromosomal regions and loci in rye. This genome sequence promises to accelerate genomic and breeding studies in rye and related cereal crops.

A distinct class of plant and animal viral proteins that disrupt mitosis by directly interrupting the mitotic entry switch Wee1-Cdc25-Cdk1.

Many animal viral proteins, e.g., Vpr of HIV-1, disrupt host mitosis by directly interrupting the mitotic entry switch Wee1-Cdc25-Cdk1. However, it is unknown whether plant viruses may use this mechanism in their pathogenesis. Here, we report that the 17K protein, encoded by barley yellow dwarf viruses and related poleroviruses, delays G2/M transition and disrupts mitosis in both host (barley) and nonhost (fission yeast, Arabidopsis thaliana, and tobacco) cells through interrupting the function of Wee1-Cdc25-CDKA/Cdc2 via direct protein-protein interactions and alteration of CDKA/Cdc2 phosphorylation. When ectopically expressed, 17K disrupts the mitosis of cultured human cells, and HIV-1 Vpr inhibits plant cell growth. Furthermore, 17K and Vpr share similar secondary structural feature and common amino acid residues required for interacting with plant CDKA. Thus, our work reveals a distinct class of mitosis regulators that are conserved between plant and animal viruses and play active roles in viral pathogenesis.

Genetic analysis of chromosomal loci affecting the content of insoluble glutenin in common wheat.

In common wheat, insoluble glutenin (IG) is an important fraction of flour glutenin macropolymers, and insoluble glutenin content (IGC) is positively associated with key end-use quality parameters. Here, we present a genetic analysis of the chromosomal loci affecting IGC with the data collected from 90 common wheat varieties cultivated in four environments. Statistical analysis showed that IGC was controlled mainly genetically and influenced by the environment. Among the major genetic components known to affect end-use quality, 1BL/1RS translocation had a significantly negative effect on IGC across all four environments. As to the different alleles of Glu-A1, -B1 and -D1 loci, Glu-A1a, Glu-B1b and Glu-D1d exhibited relatively strong positive effects on IGC in all environments. To identify new loci affecting IGC, association mapping with 1355 DArT markers was conducted. A total of 133 markers were found associated with IGC in two or more environments (P < 0.05), ten of which consistently affected IGC in all four environments. The phenotypic variance explained by the ten markers varied from 4.66% to 8.03%, and their elite alleles performed significantly better than the inferior counterparts in enhancing IGC. Among the ten markers, wPt-3743 and wPt-733835 reflected the action of Glu-D1, and wPt-664972 probably indicated the effect of Glu-A1. The other seven markers, forming three clusters on 2AL, 3BL or 7BL chromosome arms, represented newly identified genetic determinants of IGC. Our work provided novel insights into the genetic control of IGC, which may facilitate wheat end-use quality improvement through molecular breeding in the future.