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The importance of wheat yellow rust disease, caused by Puccinia striiformis f. sp. tritici (Pst), has increased substantially due to the emergence of aggressive new Pst races in the last couple of decades. In an era of escalating human populations and climate change, it is vital to understand the infection mechanism of Pst in order to develop better strategies to combat wheat yellow disease. The present study focuses on the identification of small secreted proteins (SSPs) and candidate-secreted effector proteins (CSEPs) that are used by the pathogen to support infection and control disease development. We generated de novo assembled transcriptomes of Pst collected from wheat fields in central Anatolia. We inoculated both susceptible and resistant seedlings with Pst and analyzed haustoria formation. At 10 days post-inoculation (dpi), we analyzed the transcriptomes and identified 10550 Differentially Expressed Unigenes (DEGs), of which 6072 were Pst-mapped. Among those Pst-related genes, 227 were predicted as PstSSPs. In silico characterization was performed using an approach combining the transcriptomic data and data mining results to provide a reliable list to narrow down the ever-expanding repertoire of predicted effectorome. The comprehensive analysis detected 14 Differentially Expressed Small-Secreted Proteins (DESSPs) that overlapped with the genes in available literature data to serve as the best CSEPs for experimental validation. One of the CSEPs was cloned and studied to test the reliability of the presented data. Biological assays show that the randomly selected CSEP, Unigene17495 (PSTG_10917), localizes in the chloroplast and is able to suppress cell death induced by INF1 in a Nicotiana benthamiana heterologous expression system.

Innovation toward ecofriendly plant protection products compatible with sustainable agriculture and healthy food is today strongly encouraged. Here, we assessed the biocontrol activity of three cyclic lipopeptides from Bacillus subtilis (mycosubtilin, M; surfactin, S; fengycin, F) and two mixtures (M + S and M + S + F) on wheat against Zymoseptoria tritici , the main pathogen on this crop. Foliar application of these biomolecules at a 100-mg L −1 concentration on the wheat cultivars Dinosor and Alixan, 2 days before fungal inoculation, provided significant reductions of disease severity. The best protection levels were recorded with the M-containing formulations (up to 82% disease reduction with M + S on Dinosor), while S and F treatments resulted in lower but significant disease reductions. In vitro and in planta investigations revealed that M-based formulations inhibit fungal growth, with half-maximal inhibitory concentrations of 1.4 mg L −1 for both M and M + S and 4.5 mg L −1 for M + S + F, thus revealing that the observed efficacy of these products may rely mainly on antifungal property. By contrast, S and F had no direct activity on the pathogen, hence suggesting that these lipopeptides act on wheat against Z. tritici as resistance inducers rather than as biofungicides. This study highlighted the efficacy of several lipopeptides from B. subtilis to biocontrol Z. tritici through likely distinct and biomolecule-dependent modes of action.

Plant pathogens pose a major threat to crop productivity. Typically, phytopathogens exploit plants’ susceptibility (S) genes to facilitate their proliferation. Disrupting these S genes may interfere with the compatibility between the host and the pathogens and consequently provide broad-spectrum and durable disease resistance. In the past, genetic manipulation of such S genes has been shown to confer disease resistance in various economically important crops. Recent studies have accomplished this task in a transgene-free system using new genome editing tools, including clustered regularly interspaced palindromic repeats (CRISPR). In this Opinion article, we focus on the use of genome editing to target S genes for the development of transgene-free and durable disease-resistant crop varieties. CRISPR has emerged as a revolutionary tool for plant genome editing. Although developed recently, it has been established in several important plant species, including rice, wheat, and maize, to introduce agronomically important traits such as heat/cold tolerance, disease resistance, herbicide tolerance, and yield improvement. Transgene-free methods are being introduced in CRISPR-mediated plant genome editing, such as segregating out transgenes, delivering the ribonucleoprotein complex of Cas9 and gRNA through particle bombardment or using a protoplast system, and using viral vectors for editing germline cells. Targeting susceptibility (S) genes using CRISPR methodologies offers new frontiers to break molecular plant–microbe compatibility and introducing durable pathogen resistance.

Disease resistance genes encoding nucleotide-binding and leucine-rich repeat (NLR) intracellular immune receptor proteins detect pathogens by the presence of pathogen effectors. Plant genomes typically contain hundreds of NLR-encoding genes. The availability of the hexaploid wheat (Triticum aestivum) cultivar Chinese Spring reference genome allows a detailed study of its NLR complement. However, low NLR expression and high intrafamily sequence homology hinder their accurate annotation. Here, we developed NLR-Annotator, a software tool for in silico NLR identification independent of transcript support. Although developed for wheat, we demonstrate the universal applicability of NLR-Annotator across diverse plant taxa. We applied our tool to wheat and combined it with a transcript-validated subset of genes from the reference gene annotation to characterize the structure, phylogeny, and expression profile of the NLR gene family. We detected 3,400 full-length NLR loci, of which 1,560 were confirmed as expressed genes with intact open reading frames. NLRs with integrated domains mostly group in specific subclades. Members of another subclade predominantly locate in close physical proximity to NLRs carrying integrated domains, suggesting a paired helper function. Most NLRs (88%) display low basal expression (in the lower 10 percentile of transcripts). In young leaves subjected to biotic stress, we found up-regulation of 266 of the NLRs. To illustrate the utility of our tool for the positional cloning of resistance genes, we estimated the number of NLR genes within the intervals of mapped rust resistance genes. Our study will support the identification of functional resistance genes in wheat to accelerate the breeding and engineering of disease-resistant varieties.

Powdery mildew is one of the most devastating diseases of wheat. To date, few powdery mildew resistance genes have been cloned from wheat due to the size and complexity of the wheat genome. Triticum urartu is the progenitor of the A genome of wheat and is an important source for powdery mildew resistance genes. Using molecular markers designed from scaffolds of the sequenced T. urartu accession and standard map-based cloning, a powdery mildew resistance locus was mapped to a 356-kb region, which contains two nucleotide-binding and leucinerich repeat domain (NB-LRR) protein-encoding genes. Virus-induced gene silencing, single-cell transient expression, and stable transformation assays demonstrated that one of these two genes, designated Pm60, confers resistance to powdery mildew. Overexpression of full-length Pm60 and two allelic variants in Nicotiana benthamiana leaves induced hypersensitive cell death response, but expression of the coiledcoil domain alone was insufficient to induce hypersensitive response. Yeast two-hybrid, bimolecular fluorescence complementation and luciferase complementation imaging assays showed that Pm60 protein interacts with its neighboring NB-containing protein, suggesting that they might be functionally related. The identification and cloning of this novel wheat powdery mildew resistance gene will facilitate breeding for disease resistance in wheat.

Disruption of susceptibility ( S ) genes in crops is an attractive breeding strategy for conferring disease resistance 1 , 2 . However, S genes are implicated in many essential biological functions and deletion of these genes typically results in undesired pleiotropic effects 1 . Loss-of-function mutations in one such S gene, Mildew resistance locus O ( MLO ), confers durable and broad-spectrum resistance to powdery mildew in various plant species 2 , 3 . However, mlo- associated resistance is also accompanied by growth penalties and yield losses 3 , 4 , thereby limiting its widespread use in agriculture. Here we describe Tamlo-R32 , a mutant with a 304-kilobase pair targeted deletion in the MLO-B1 locus of wheat that retains crop growth and yields while conferring robust powdery mildew resistance. We show that this deletion results in an altered local chromatin landscape, leading to the ectopic activation of Tonoplast monosaccharide transporter 3 ( TaTMT3B ), and that this activation alleviates growth and yield penalties associated with MLO disruption. Notably, the function of TMT3 is conserved in other plant species such as Arabidopsis thaliana . Moreover, precision genome editing facilitates the rapid introduction of this mlo resistance allele ( Tamlo-R32) into elite wheat varieties. This work demonstrates the ability to stack genetic changes to rescue growth defects caused by recessive alleles, which is critical for developing high-yielding crop varieties with robust and durable disease resistance. Tamlo-R32 , an engineered wheat mutant allele of the Mildew resistance locus O ( MLO ) gene, confers resistance to powdery mildew, retains robust wheat growth, and can be transferred to other agriculturally important wheat varieties.

Evans Lagudah and colleagues report that variation in a gene encoding a hexose transporter confers resistance to multiple pathogens in wheat. They further show that the variant protein encoded by the resistance allele exerts a dominant-negative effect by heterodimerizing with functional hexose transporters, resulting in reduced glucose uptake. As there are numerous pathogen species that cause disease and limit yields of crops, such as wheat ( Triticum aestivum ), single genes that provide resistance to multiple pathogens are valuable in crop improvement 1 , 2 . The mechanistic basis of multi-pathogen resistance is largely unknown. Here we use comparative genomics, mutagenesis and transformation to isolate the wheat Lr67 gene, which confers partial resistance to all three wheat rust pathogen species and powdery mildew. The Lr67 resistance gene encodes a predicted hexose transporter (LR67res) that differs from the susceptible form of the same protein (LR67sus) by two amino acids that are conserved in orthologous hexose transporters. Sugar uptake assays show that LR67sus, and related proteins encoded by homeoalleles, function as high-affinity glucose transporters. LR67res exerts a dominant-negative effect through heterodimerization with these functional transporters to reduce glucose uptake. Alterations in hexose transport in infected leaves may explain its ability to reduce the growth of multiple biotrophic pathogen species.

The cloning of agronomically important genes from large, complex crop genomes remains challenging. Here we generate a 14.7 gigabase chromosome-scale assembly of the South African bread wheat ( Triticum aestivum ) cultivar Kariega by combining high-fidelity long reads, optical mapping and chromosome conformation capture. The resulting assembly is an order of magnitude more contiguous than previous wheat assemblies. Kariega shows durable resistance to the devastating fungal stripe rust disease 1 . We identified the race-specific disease resistance gene Yr27 , which encodes an intracellular immune receptor, to be a major contributor to this resistance. Yr27 is allelic to the leaf rust resistance gene Lr13 ; the Yr27 and Lr13 proteins show 97% sequence identity 2 , 3 . Our results demonstrate the feasibility of generating chromosome-scale wheat assemblies to clone genes, and exemplify that highly similar alleles of a single-copy gene can confer resistance to different pathogens, which might provide a basis for engineering Yr27 alleles with multiple recognition specificities in the future. Chromosome-scale genome assembly of the South African bread wheat ( Triticum aestivum ) cultivar Kariega facilitates the cloning of the stripe rust resistance gene Yr27 .

Key messageNew powdery mildew resistance gene Pm68 was found in the terminal region of chromosome 2BS of Greek durum wheat TRI 1796. The co-segregated molecular markers could be used for MAS.Durum wheat (Triticum turgidum L. var. durum Desf.) is not only an important cereal crop for pasta making, but also a genetic resource for common wheat improvement. In the present study, a Greek durum wheat TRI 1796 was found to confer high resistance to all 22 tested isolates of Blumeria graminis f. sp. tritici (Bgt). Inheritance study on the F1 plants and the F2 population derived from the cross TRI 1796/PI 584832 revealed that the resistance in TRI 1796 was controlled by a single dominant gene, herein designated Pm68. Using the bulked segregant RNA-Seq (BSR-Seq) analysis combined with molecular analysis, Pm68 was mapped to the terminal part of the short arm of chromosome 2B and flanked by markers Xdw04 and Xdw12/Xdw13 with genetic distances of 0.22 cM each. According to the reference genome of durum wheat cv. Svevo, the corresponding physical region spanned the Pm68 locus was about 1.78-Mb, in which a number of disease resistance-related genes were annotated. This study reports the new powdery mildew resistance gene Pm68 that would be a valuable resource for improvement of both common wheat and durum wheat. The co-segregated markers (Xdw05–Xdw11) developed here would be useful tools for marker-assisted selection (MAS) in breeding.

Plant growth promoting rhizobacteria (PGPR) hold promising future for sustainable agriculture. Here, we demonstrate a carotenoid producing halotolerant PGPR Dietzia natronolimnaea STR1 protecting wheat plants from salt stress by modulating the transcriptional machinery responsible for salinity tolerance in plants. The expression studies confirmed the involvement of ABA-signalling cascade, as TaABARE and TaOPR1 were upregulated in PGPR inoculated plants leading to induction of TaMYB and TaWRKY expression followed by stimulation of expression of a plethora of stress related genes. Enhanced expression of TaST , a salt stress-induced gene, associated with promoting salinity tolerance was observed in PGPR inoculated plants in comparison to uninoculated control plants. Expression of SOS pathway related genes ( SOS1 and SOS4 ) was modulated in PGPR-applied wheat shoots and root systems. Tissue-specific responses of ion transporters TaNHX1 , TaHAK , and TaHKT1 , were observed in PGPR-inoculated plants. The enhanced gene expression of various antioxidant enzymes such as APX , MnSOD , CAT , POD , GPX and GR and higher proline content in PGPR-inoculated wheat plants contributed to increased tolerance to salinity stress. Overall, these results indicate that halotolerant PGPR-mediated salinity tolerance is a complex phenomenon that involves modulation of ABA-signalling, SOS pathway, ion transporters and antioxidant machinery.