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The complicated interplay of plant–pathogen interactions occurs on multiple levels as pathogens evolve to constantly evade the immune responses of their hosts. Many economically important crops fall victim to filamentous pathogens that produce small proteins called effectors to manipulate the host and aid infection/colonization. Understanding the effector repertoires of pathogens is facilitating an increased understanding of the molecular mechanisms underlying virulence as well as guiding the development of disease control strategies. The purpose of this review is to give a chronological perspective on the evolution of the methodologies used in effector discovery from physical isolation and in silico predictions, to functional characterization of the effectors of filamentous plant pathogens and identification of their host targets. The chronological progression of methodologies used in effector discovery; from physical isolation and in silico predictions, to functional characterization and host target identification.

CYP51 encodes the cytochrome P450 sterol 14α-demethylase, an enzyme essential for sterol biosynthesis and the target of azole fungicides. In Fusarium species, including pathogens of humans and plants, three CYP51 paralogues have been identified with one unique to the genus. Currently, the functions of these three genes and the rationale for their conservation within the genus Fusarium are unknown. Three Fusarium graminearum CYP51s (FgCYP51s) were heterologously expressed in Saccharomyces cerevisiae. Single and double FgCYP51 deletion mutants were generated and the functions of the FgCYP51s were characterized in vitro and in planta. FgCYP51A and FgCYP51B can complement yeast CYP51 function, whereas FgCYP51C cannot. FgCYP51A deletion increases the sensitivity of F. graminearum to the tested azoles. In ΔFgCYP51B and ΔFgCYP51BC mutants, ascospore formation is blocked, and eburicol and two additional 14-methylated sterols accumulate. FgCYP51C deletion reduces virulence on host wheat ears. FgCYP51B encodes the enzyme primarily responsible for sterol 14α-demethylation, and plays an essential role in ascospore formation. FgCYP51A encodes an additional sterol 14α-demethylase, induced on ergosterol depletion and responsible for the intrinsic variation in azole sensitivity. FgCYP51C does not encode a sterol 14α-demethylase, but is required for full virulence on host wheat ears. This is the first example of the functional diversification of a fungal CYP51.

Deployment of fast-evolving disease-resistance genes is one of the most successful strategies used by plants to fend off pathogens 1 , 2 . In gene-for-gene relationships, most cloned disease-resistance genes encode intracellular nucleotide-binding leucine-rich-repeat proteins (NLRs) recognizing pathogen-secreted isolate-specific avirulence (Avr) effectors delivered to the host cytoplasm 3 , 4 . This process often triggers a localized hypersensitive response, which halts further disease development 5 . Here we report the map-based cloning of the wheat Stb6 gene and demonstrate that it encodes a conserved wall-associated receptor kinase (WAK)-like protein, which detects the presence of a matching apoplastic effector 6 – 8 and confers pathogen resistance without a hypersensitive response 9 . This report demonstrates gene-for-gene disease resistance controlled by this class of proteins in plants. Moreover, Stb6 is, to our knowledge, the first cloned gene specifying resistance to Zymoseptoria tritici , an important foliar fungal pathogen affecting wheat and causing economically damaging septoria tritici blotch (STB) disease 10 – 12 . The authors report map-based cloning of the wheat Stb6 gene, which encodes a conserved wall-associated receptor kinase (WAK)-like protein. Stb6 confers gene-for-gene disease resistance to fungal pathogen Zymoseptoria tritici by recognition of a matching pathogen effector.

Rapid and cost-effective virus-derived transient expression systems for plants are invaluable in elucidating gene function and are particularly useful in plant species for which transformation-based methods are unavailable or are too time and labor demanding, such as wheat (Triticum aestivum) and maize (Zea mays). The virus-mediated overexpression (VOX) vectors based on Barley stripe mosaic virus and Wheat streak mosaic virus described previously for these species are incapable of expressing free recombinant proteins of more than 150 to 250 amino acids, are not suited for high-throughput screens, and have other limitations. In this study, we report the development of a VOX vector based on a monopartite single-stranded positive sense RNA virus, Foxtail mosaic virus (genus Potexvirus). In this vector, PV101, the gene of interest was inserted downstream of the duplicated subgenomic promoter of the viral coat protein gene, and the corresponding protein was expressed in its free form. The vector allowed the expression of a 239-amino acid-long GFP in both virus-inoculated and upper uninoculated (systemic) leaves of wheat and maize and directed the systemic expression of a larger approximately 600-amino acid protein, GUSPlus, in maize. Moreover, we demonstrated that PV101 can be used for in planta expression and functional analysis of apoplastic pathogen effector proteins such as the host-specific toxin ToxA of Parastagonospora nodorum. Therefore, this VOX vector opens possibilities for functional genomics studies in two important cereal crops.

The fungus Fusarium graminearum forms an intimate association with the host species wheat whilst infecting the floral tissues at anthesis. During the prolonged latent period of infection, extracellular communication between live pathogen and host cells must occur, implying a role for secreted fungal proteins. The wheat cells in contact with fungal hyphae subsequently die and intracellular hyphal colonisation results in the development of visible disease symptoms. Since the original genome annotation analysis was done in 2007, which predicted the secretome using TargetP, the F. graminearum gene call has changed considerably through the combined efforts of the BROAD and MIPS institutes. As a result of the modifications to the genome and the recent findings that suggested a role for secreted proteins in virulence, the F. graminearum secretome was revisited. In the current study, a refined F. graminearum secretome was predicted by combining several bioinformatic approaches. This strategy increased the probability of identifying truly secreted proteins. A secretome of 574 proteins was predicted of which 99% was supported by transcriptional evidence. The function of the annotated and unannotated secreted proteins was explored. The potential role(s) of the annotated proteins including, putative enzymes, phytotoxins and antifungals are discussed. Characterisation of the unannotated proteins included the analysis of Pfam domains and features associated with known fungal effectors, for example, small size, cysteine-rich and containing internal amino acid repeats. A comprehensive comparative genomic analysis involving 57 fungal and oomycete genomes revealed that only a small number of the predicted F. graminearum secreted proteins can be considered to be either species or sequenced strain specific.

Fusarium head blight disease on small‐grain cereals is primarily caused by the ascomycete fungal pathogen Fusarium graminearum. Infection of floral spike tissues is characterized by the biosynthesis and secretion of potent trichothecene mycotoxins, of which deoxynivalenol (DON) is widely reported due to its negative impacts on grain quality and consumer safety. The TRI5 gene encodes an essential enzyme in the DON biosynthesis pathway and the single gene deletion mutant, ΔTri5, is widely reported to restrict disease progression to the inoculated spikelet. In this study, we present novel bioimaging evidence revealing that DON facilitates the traversal of the cell wall through plasmodesmata, a process essential for successful colonization of host tissue. Chemical complementation of ΔTri5 did not restore macro‐ or microscopic phenotypes, indicating that DON secretion is tightly regulated both spatially and temporally. A comparative qualitative and quantitative morphological cellular analysis revealed infections had no impact on plant cell wall thickness. Immunolabelling of callose at plasmodesmata during infection indicates that DON can increase deposits when applied exogenously but is reduced when F. graminearum hyphae are present. This study highlights the complexity of the interconnected roles of mycotoxin production, cell wall architecture and plasmodesmata in this highly specialized interaction. The trichothecene mycotoxin deoxynivalenol (DON) dampens host immune responses and facilitates cell‐to‐cell invasion through plasmodesmatal pit fields in wheat floral tissues.

A data set of promoter and 5′UTR sequences of homoeo‐alleles of 459 wheat genes that contribute to agriculturally important traits in 95 ancestral and commercial wheat cultivars is presented here. The high‐stringency myBaits technology used made individual capture of homoeo‐allele promoters possible, which is reported here for the first time. Promoters of most genes are remarkably conserved across the 83 hexaploid cultivars used with <7 haplotypes per promoter and 21% being identical to the reference Chinese Spring. InDels and many high‐confidence SNPs are located within predicted plant transcription factor binding sites, potentially changing gene expression. Most haplotypes found in the Watkins landraces and a few haplotypes found in Triticum monococcum, germplasms hitherto not thought to have been used in modern wheat breeding, are already found in many commercial hexaploid wheats. The full data set which is useful for genomic and gene function studies and wheat breeding is available at https://rrescloud.rothamsted.ac.uk/index.php/s/DMCFDu5iAGTl50u/authenticate.

Background The soil dwelling saprotrophic non-pathogenic fungus Fusarium venenatum , routinely used in the commercial fermentation industry, is phylogenetically closely related to the globally important cereal and non-cereal infecting pathogen F. graminearum . This study aimed to sequence, assemble and annotate the F. venenatum (strain A3/5) genome, and compare this genome with F. graminearum . Results Using shotgun sequencing, a 38,660,329 bp  F. venenatum genome was assembled into four chromosomes, and a 78,618 bp mitochondrial genome. In comparison to F. graminearum , the predicted gene count of 13,946 was slightly lower. The F. venenatum centromeres were found to be 25% smaller compared to F. graminearum . Chromosome length was 2.8% greater in F. venenatum, primarily due to an increased abundance of repetitive elements and transposons, but not transposon diversity. On chromosome 3 a major sequence rearrangement was found, but its overall gene content was relatively unchanged. Unlike homothallic F. graminearum , heterothallic F. venenatum possessed the MAT1–1 type locus, but lacked the MAT1–2 locus. The F. venenatum genome has the type A trichothecene mycotoxin TRI5 cluster, whereas F. graminearum has type B. From the F. venenatum gene set, 786 predicted proteins were species-specific versus NCBI. The annotated F. venenatum genome was predicted to possess more genes coding for hydrolytic enzymes and species - specific genes involved in the breakdown of polysaccharides than F. graminearum . Comparison of the two genomes reduced the previously defined F. graminearum- specific gene set from 741 to 692 genes. A comparison of the F. graminearum versus F. venenatum proteomes identified 15 putative secondary metabolite gene clusters (SMC), 109 secreted proteins and 38 candidate effectors not found in F. venenatum . Five of the 15 F. graminearum-specific SMCs that were either absent or highly divergent in the F. venenatum genome showed increased in planta expression. In addition, two predicted F. graminearum transcription factors previously shown to be required for fungal virulence on wheat plants were absent or exhibited high sequence divergence. Conclusions This study identifies differences between the F. venenatum and F. graminearum genomes that may contribute to contrasting lifestyles, and highlights the repertoire of F. graminearum -specific candidate genes and SMCs potentially required for pathogenesis.

Fusarium Head Blight (FHB) is the number one floral disease of cereals and poses a serious health hazard by contaminating grain with the harmful mycotoxin deoxynivalenol (DON). Fungi adapt to fluctuations in their environment, coordinating development and metabolism accordingly. G-protein coupled receptors (GPCRs) communicate changes in the environment to intracellular G-proteins that direct the appropriate biological response, suggesting that fungal GPCR signalling may be key to virulence. Here we describe the expansion of non-classical GPCRs in the FHB causing pathogen, Fusarium graminearum, and show that class X receptors are highly expressed during wheat infection. We identify class X receptors that are required for FHB disease on wheat, and show that the absence of a GPCR can cause an enhanced host response that restricts the progression of infection. Specific receptor sub-domains are required for virulence. These non-classical receptors physically interact with intracellular G-proteins and are therefore bona fide GPCRs. Disrupting a class X receptor is shown to dysregulate the transcriptional coordination of virulence traits during infection. This amounts to enhanced wheat defensive responses, including chitinase and plant cell wall biosynthesis, resulting in apoplastic and vascular occlusions that impede infection. Our results show that GPCR signalling is important to FHB disease establishment.

Wheat is an economically, socially, and nutritionally important crop, however, aphid infestation can often reduce wheat yield through feeding and virus transmission. Through field phenotyping, we investigated aphid resistance in ancestral wheat Triticum monococcum (L.). Aphid ( Rhopalosiphum padi (L.) , Sitobion avenae (F.) and Metopolophium dirhodum (Wlk.)) populations and natural enemy presence (parasitised mummified aphids, ladybird adults and larvae and lacewing eggs and larvae) on two naturally susceptible wheat varieties, Triticum aestivum (L.) var. Solstice and T. monococcum MDR037, and three potentially resistant genotypes T. monococcum MDR657, MDR045 and MDR049 were monitored across three years of field trials. Triticum monococcum MDR045 and MDR049 had smaller aphid populations, whereas MDR657 showed no resistance. Overall, natural enemy presence was positively correlated with aphid populations; however, MDR049 had similar natural enemy presence to MDR037 which is susceptible to aphid infestation. It is hypothesised that alongside reducing aphid population growth, MDR049 also confers indirect resistance by attracting natural enemies. The observed resistance to aphids in MDR045 and MDR049 has strong potential for introgression into commercial wheat varieties, which could have an important role in Integrated Pest Management strategies to reduce aphid populations and virus transmission.