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Summary: Parastagonospora nodorum is an economically important necrotrophic fungal pathogen of wheat. Parastagonospora nodorum secretes necrotrophic effectors that target wheat susceptibility genes to induce programmed cell death (PCD). In this study, we cloned and functionally validated SnTox5 and characterized its role in pathogenesis.We used whole genome sequencing, genome‐wide association study (GWAS) mapping, CRISPR‐Cas9‐based gene disruption, gain‐of‐function transformation, quantitative trait locus (QTL) analysis, haplotype and isoform analysis, protein modeling, quantitative PCR, and laser confocal microscopy to validate SnTox5 and functionally characterize SnTox5.SnTox5 is a mature 16.26 kDa protein with high structural similarity to SnTox3. Wild‐type and mutant P. nodorum strains and wheat genotypes of SnTox5 and Snn5, respectively, were used to show that SnTox5 not only targets Snn5 to induce PCD but also facilitates the colonization of the mesophyll layer even in the absence of Snn5.Here we show that SnTox5 facilitates the efficient colonization of the mesophyll tissue and elicits PCD specific to host lines carrying Snn5. The homology to SnTox3 and the ability of SnTox5 to facilitate the colonizing of the mesophyll also suggest a role in the suppression of host defense before PCD induction. See also the Commentary on this article by Kanyuka et al., 233: 11–14. [ABSTRACT FROM AUTHOR]

Summary: Parastagonospora nodorum is a fungal pathogen of wheat. As a necrotrophic specialist, it deploys effector proteins that target dominant host susceptibility genes to elicit programmed cell death (PCD). Here we identify and functionally validate the effector targeting the host susceptibility genes Snn2, Snn6 and Snn7.We utilized whole‐genome sequencing, association mapping, gene‐disrupted mutants, gain‐of‐function transformants, virulence assays, bioinformatics and quantitative PCR to characterize these interactions.A single proteinaceous effector, SnTox267, targeted Snn2, Snn6 and Snn7 to trigger PCD. Snn2 and Snn6 functioned cooperatively to trigger PCD in a light‐dependent pathway, whereas Snn7‐mediated PCD functioned in a light‐independent pathway. Isolates harboring 20 SnTox267 protein isoforms quantitatively varied in virulence. The diversity and distribution of isoforms varied between populations, indicating adaptation to local selection pressures. SnTox267 deletion resulted in the upregulation of effector genes SnToxA, SnTox1 and SnTox3.We validated a novel effector operating in an inverse‐gene‐for‐gene manner to target three genetically distinct host susceptibility genes and elicit PCD. The discovery of the complementary gene action of Snn2 and Snn6 indicates their potential function in a guard or decoy model. Additionally, differences in light dependency in the elicited pathways and upregulation of unlinked effectors sheds new light onto a complex fungal necrotroph–host interaction. See also the Commentary on this article by Kanyuka et al., 233: 11–14. [ABSTRACT FROM AUTHOR]

Xanthomonas translucens is a group of gram‐negative bacteria that can cause important diseases in cereal crops and forage grasses. Different pathovars have been defined according to their host ranges, and molecular and biochemical characteristics. Pathovars have been placed into two major groups: translucens and graminis. The translucens group contains the pathovars causing bacterial leaf streak (BLS) on cereal crops such as wheat, barley, triticale, rye, and oat. In recent years, BLS has re‐emerged as a major problem for many wheat‐ and barley‐producing areas worldwide. The biology of the pathogens and the host–pathogen interactions in cereal BLS diseases were poorly understood. However, recent genome sequence data have provided an insight into the bacterial phylogeny and identification and pathogenicity/virulence. Furthermore, identification of sources of resistance to BLS and mapping of the resistance genes have been initiated. Taxonomy: Kingdom Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; Species X. translucens; translucens group pathovars: undulosa, translucens, cerealis, hordei, and secalis; graminis group pathovars: arrhenatheri, graminis, poae, phlei; newly established pathovar: pistaciae. Host range: X. translucens mainly infects plant species in the Poaceae with the translucens group on cereal crop species and the graminis group on forage grass species. However, some strains have been isolated from, and are able to infect, ornamental asparagus and pistachio trees. Most pathovars have a narrow host range, while a few can infect a broad range of hosts. Genome: The complete genome sequence is available for two X. translucens pv. undulosa strains and one pv. translucens strain. A draft genome sequence is also available for at least one strain from each pathovar. The X. translucens pv. undulosa strain Xt4699 was the first to have its complete genome sequenced, which consists of 4,561,137 bp with total GC content approximately at 68% and 3,528 predicted genes. Virulence mechanisms: Like most xanthomonads, X. translucens utilizes a type III secretion system (T3SS) to deliver a suite of T3SS effectors (T3Es) inside plant cells. Transcription activator‐like effectors, a special group of T3Es, have been identified in most of the X. translucens genomes, some of which have been implicated in virulence. Genetic factors determining host range virulence have also been identified. [ABSTRACT FROM AUTHOR]

SnTox1 induces programmed cell death and the up-regulation of pathogenesis-related genes including chitinases. Additionally, SnTox1 has structural homology to several plant chitin-binding proteins., Therefore, we evaluated SnTox1 for chitin binding and localization. We transformed an avirulent strain of Parastagonospora nodorum as well as three nonpathogens of wheat ( Triticum aestivum), including a necrotrophic pathogen of barley, a hemibiotrophic pathogen of sugar beet and a saprotroph, to evaluate the role of SnTox1 in infection and in protection from wheat chitinases., SnTox1 bound chitin and an SnTox1-green fluorescent fusion protein localized to the mycelial cell wall. Purified SnTox1 induced necrosis in the absence of the pathogen when sprayed on the leaf surface and appeared to remain on the leaf surface while inducing both epidermal and mesophyll cell death. SnTox1 protected the different fungi from chitinase degradation. SnTox1 was sufficient to change the host range of a necrotrophic pathogen but not a hemibiotroph or saprotroph., Collectively, this work shows that SnTox1 probably interacts with a receptor on the outside of the cell to induce cell death to acquire nutrients, but SnTox1 accomplishes a second role in that it protects against one aspect of the defense response, namely the effects of wheat chitinases. [ABSTRACT FROM AUTHOR]

Barley net form net blotch ( NFNB), caused by the necrotrophic fungus Pyrenophora teres f. teres, is a destructive foliar disease in barley-growing regions worldwide. Little is known about the genetic and molecular basis of this pathosystem. Here, we identified a small secreted proteinaceous necrotrophic effector ( NE), designated PttNE1, from intercellular wash fluids of the susceptible barley line Hector after inoculation with P. teres f. teres isolate 0-1. Using a barley recombinant inbred line (RIL) population developed from a cross between the sensitive/susceptible line Hector and the insensitive/resistant line NDB 112 ( HN population), sensitivity to PttNE1, which we have named SPN1, mapped to a common resistance/susceptibility region on barley chromosome 6H. PttNE1- SPN1 interaction accounted for 31% of the disease variation when the HN population was inoculated with the 0-1 isolate. Strong accumulation of hydrogen peroxide and increased levels of electrolyte leakage were associated with the susceptible reaction, but not the resistant reaction. In addition, the HN RIL population was evaluated for its reactions to 10 geographically diverse P. teres f. teres isolates. Quantitative trait locus ( QTL) mapping led to the identification of at least 10 genomic regions associated with disease, with chromosomes 3H and 6H harbouring major QTLs for resistance/susceptibility. SPN1 was associated with all the 6H QTLs, except one. Collectively, this information indicates that the barley- P. teres f. teres pathosystem follows, at least partially, an NE-triggered susceptibility ( NETS) model that has been described in other necrotrophic fungal disease systems, especially in the Dothideomycete class of fungi. [ABSTRACT FROM AUTHOR]

A high-throughput RNA-mediated gene silencing system was developed for Cochliobolus sativus (anamorph: Bipolaris sorokiniana), the causal agent of spot blotch, common root rot and black point in barley and wheat. The green fluorescent protein gene ( GFP) and the proteinaceous host-selective toxin gene ( ToxA) were first introduced into C. sativus via the polyethylene glycol (PEG)-mediated transformation method. Transformants with a high level of expression of GFP or ToxA were generated. A silencing vector (pSGate1) based on the Gateway cloning system was developed and used to construct RNA interference (RNAi) vectors. Silencing of GFP and ToxA in the transformants was demonstrated by transformation with the RNAi construct expressing hairpin RNA (hpRNA) of the target gene. The polyketide synthase gene ( CsPKS1), involved in melanin biosynthesis pathways in C. sativus, was also targeted by transformation with the RNAi vector (pSGate1- CsPKS1) encoding hpRNA of the CsPKS1 gene. The transformants with pSGate1- CsPKS1 exhibited an albino phenotype or reduced melanization, suggesting effective silencing of the endogenous CsPKS1 in C. sativus. Sectors exhibiting the wild-type phenotype of the fungus appeared in some of the CsPKS1-silenced transformants after subcultures as a result of inactivation or deletions of the RNAi transgene. The gene silencing system established provides a useful tool for functional genomics studies in C. sativus and other filamentous fungi. [ABSTRACT FROM AUTHOR]

Pyrenophora teres, causal agent of net blotch of barley, exists in two forms, designated P. teres f. teres and P. teres f. maculata, which induce net form net blotch (NFNB) and spot form net blotch (SFNB), respectively. Significantly more work has been performed on the net form than on the spot form although recent activity in spot form research has increased because of epidemics of SFNB in barley-producing regions. Genetic studies have demonstrated that NFNB resistance in barley is present in both dominant and recessive forms, and that resistance/susceptibility to both forms can be conferred by major genes, although minor quantitative trait loci have also been identified. Early work on the virulence of the pathogen showed toxin effector production to be important in disease induction by both forms of pathogen. Since then, several laboratories have investigated effectors of virulence and avirulence, and both forms are complex in their interaction with the host. Here, we assemble recent information from the literature that describes both forms of this important pathogen and includes reports describing the host-pathogen interaction with barley. We also include preliminary findings from a genome sequence survey. Pyrenophora teres Drechs. Kingdom Fungi; Phylum Ascomycota; Subphylum Pezizomycotina; Class Dothideomycete; Order Pleosporales; Family Pleosporaceae; Genus Pyrenophora, form teres and form maculata. To date, no clear morphological or life cycle differences between the two forms of P. teres have been identified, and therefore they are described collectively. Towards the end of the growing season, the fungus produces dark, globosely shaped pseudothecia, about 1-2 mm in diameter, on barley. Ascospores measuring 18-28 µm × 43-61 µm are light brown and ellipsoidal and often have three to four transverse septa and one or two longitudinal septa in the median cells. Conidiophores usually arise singly or in groups of two or three and are lightly swollen at the base. Conidia measuring 30-174 µm × 15-23 µm are smoothly cylindrical and straight, round at both ends, subhyaline to yellowish brown, often with four to six pseudosepta. Morphologically, P. teres f. teres and P. teres f. maculata are indistinguishable. Comprehensive work on the host range of P. teres f. teres has been performed; however, little information on the host range of P. teres f. maculata is available. Hordeum vulgare and H. vulgare ssp. spontaneum are considered to be the primary hosts for P. teres. However, natural infection by P. teres has been observed in other wild Hordeum species and related species from the genera Bromus, Avena and Triticum, including H. marinum, H. murinum, H. brachyantherum, H. distichon, H. hystrix, B. diandrus, A. fatua, A. sativa and T. aestivum (, Rev. Plant Pathol. 52:269-290). In artificial inoculation experiments under field conditions, P. teres f. teres has been shown to infect a wide range of gramineous species in the genera Agropyron, Brachypodium, Elymus, Cynodon, Deschampsia, Hordelymus and Stipa (Brown et al., 1993, Plant Dis. 77:942-947). Additionally, 43 gramineous species were used in a growth chamber study and at least one of the P. teres f. teres isolates used was able to infect 28 of the 43 species tested. However, of these 28 species, 14 exhibited weak type 1 or 2 reactions on the NFNB 1-10 scale (). These reaction types are small pin-point lesions and could possibly be interpreted as nonhost reactions. In addition, the P. teres f. teres host range was investigated under field conditions by artificially inoculating 95 gramineous species with naturally infected barley straw. Pyrenophora teres f. teres was re-isolated from 65 of the species when infected leaves of adult plants were incubated on nutrient agar plates; however, other than Hordeum species, only two of the 65 host species exhibited moderately susceptible or susceptible field reaction types, with most species showing small dark necrotic lesions indicative of a highly resistant response to P. teres f. teres. Although these wild species have the potential to be alternative hosts, the high level of resistance identified for most of the species makes their role as a source of primary inoculum questionable. Two types of symptom are caused by P. teres. These are net-type lesions caused by P. teres f. teres and spot-type lesions caused by P. teres f. maculata. The net-like symptom, for which the disease was originally named, has characteristic narrow, dark-brown, longitudinal and transverse striations on infected leaves. The spot form symptom consists of dark-brown, circular to elliptical lesions surrounded by a chlorotic or necrotic halo of varying width. [ABSTRACT FROM AUTHOR]

The pathogen Stagonospora nodorum produces multiple effectors, also known as host-selective toxins (HSTs), that interact with corresponding host sensitivity genes in an inverse gene-for-gene manner to cause the disease Stagonospora nodorum blotch (SNB) in wheat. In this study, a sensitivity gene was identified in Aegilops tauschii, the diploid D-genome donor of common wheat. The gene was mapped to the short arm of chromosome 5D and mediated recognition of the effector SnTox3, which was previously shown to be recognized by the wheat gene Snn3 on chromosome arm 5BS. Comparative mapping suggested that Snn3 and the gene on 5DS are probably homoeologous and derived from a common ancestor. Therefore, we propose to designate these genes as Snn3-B1 and Snn3-D1, respectively. Compatible Snn3-D1-SnTox3 interactions resulted in more severe necrosis in both effector infiltration and spore inoculation experiments than compatible Snn3-B1-SnTox3 interactions, indicating that Snn3-B1 and Snn3-D1 may have different affinities in SnTox3 recognition or signal transduction. Wheat bin-mapped expressed sequence tags and good levels of collinearity among the wheat Snn3 regions, rice ( Oryza sativa), and Brachypodium distachyon were exploited for saturation and fine mapping of the Snn3-D1 locus. Markers delineating the Snn3-D1 locus to a 1.4 cM interval will be useful for initiating positional cloning. Further characterization of how these homoeologous genes mediate recognition of the same pathogen effector should enhance understanding of host manipulation by necrotrophic pathogens in causing disease. [ABSTRACT FROM AUTHOR]