Evolution, diversity, and function of the disease susceptibility gene Snn1 in wheat.

Septoria nodorum blotch (SNB), caused by Parastagonospora nodorum, is a disease of durum and common wheat initiated by the recognition of pathogen-produced necrotrophic effectors (NEs) by specific wheat genes. The wheat gene Snn1 was previously cloned, and it encodes a wall-associated kinase that directly interacts with the NE SnTox1 leading to programmed cell death and ultimately the development of SNB. Here, sequence analysis of Snn1 from 114 accessions including diploid, tetraploid, and hexaploid wheat species revealed that some wheat lines possess two copies of Snn1 (designated Snn1-B1 and Snn1-B2) approximately 120 kb apart. Snn1-B2 evolved relatively recently as a paralog of Snn1-B1, and both genes have undergone diversifying selection. Three point mutations associated with the formation of the first SnTox1-sensitive Snn1-B1 allele from a primitive wild wheat were identified. Four subsequent and independent SNPs, three in Snn1-B1 and one in Snn1-B2, converted the sensitive alleles to insensitive forms. Protein modeling indicated these four mutations could abolish Snn1-SnTox1 compatibility either through destabilization of the Snn1 protein or direct disruption of the protein-protein interaction. A high-throughput marker was developed for the absent allele of Snn1, and it was 100% accurate at predicting SnTox1-insensitive lines in both durum and spring wheat. Results of this study increase our understanding of the evolution, diversity, and function of Snn1-B1 and Snn1-B2 genes and will be useful for marker-assisted elimination of these genes for better host resistance.

Profiling the Pyrenophora tritici-repentis secretome: The Pf2 transcription factor regulates the secretion of the effector proteins ToxA and ToxB.

The global wheat disease tan spot is caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) which secretes necrotrophic effectors to facilitate host plant colonization. We previously reported a role of the Zn2 Cys6 binuclear cluster transcription factor Pf2 in the regulation of the Ptr effector ToxA. Here, we show that Pf2 is also a positive regulator of ToxB, via targeted deletion of PtrPf2 which resulted in reduced ToxB expression and defects in conidiation and pathogenicity. To further investigate the function of Ptr Pf2 in regulating protein secretion, the secretome profiles of two Δptrpf2 mutants of two Ptr races (races 1 and 5) were evaluated using a SWATH-mass spectrometry (MS) quantitative approach. Analysis of the secretomes of the Δptrpf2 mutants from in vitro culture filtrate identified more than 500 secreted proteins, with 25% unique to each race. Of the identified proteins, less than 6% were significantly differentially regulated by Ptr Pf2. Among the downregulated proteins were ToxA and ToxB, specific to race 1 and race 5 respectively, demonstrating the role of Ptr Pf2 as a positive regulator of both effectors. Significant motif sequences identified in both ToxA and ToxB putative promoter regions were further explored via GFP reporter assays.

The Velvet transcription factor PnVeA regulates necrotrophic effectors and secondary metabolism in the wheat pathogen Parastagonospora nodorum.

The fungus Parastagonospora nodorum causes septoria nodorum blotch on wheat. The role of the fungal Velvet-family transcription factor VeA in P. nodorum development and virulence was investigated here. Deletion of the P. nodorum VeA ortholog, PnVeA, resulted in growth abnormalities including pigmentation, abolished asexual sporulation and highly reduced virulence on wheat. Comparative RNA-Seq and RT-PCR analyses revealed that the deletion of PnVeA also decoupled the expression of major necrotrophic effector genes. In addition, the deletion of PnVeA resulted in an up-regulation of four predicted secondary metabolite (SM) gene clusters. Using liquid-chromatography mass-spectrometry, it was observed that one of the SM gene clusters led to an accumulation of the mycotoxin alternariol. PnVeA is essential for asexual sporulation, full virulence, secondary metabolism and necrotrophic effector regulation.

A New ToxA Haplotype in the Wheat Fungal Pathogen Bipolaris sorokiniana.

The necrotrophic effector ToxA is a well-studied virulence factor produced by several fungal necrotrophs. Initially cloned from the wheat tan spot pathogen Pyrenophora tritici-repentis in 1996, ToxA was found almost a decade later in another fungal pathogen, Parastagonospora nodorum, and its sister species, Parastagonospora pseudonodorum. In 2018, ToxA was detected in a third wheat fungal pathogenic species, Bipolaris sorokiniana, which causes spot blotch disease. However, unlike the case with P. tritici-repentis and P. nodorum, the ToxA in B. sorokiniana has only been investigated in recent years. In this report, five Australian B. sorokiniana isolates were assessed for the presence of ToxA. Four isolates were found to contain ToxA. While one isolate harbored the previously reported ToxA haplotype sequence (ToxA19), three isolates contain a different haplotype, designated herein as ToxA25, which has a nonsynonymous mutation resulting in an amino acid change of glycine to arginine at position 168. Both B. sorokiniana ToxA isoforms, when heterologously expressed in Escherichia coli, exhibited the classic ToxA necrosis-inducing activity on ToxA-sensitive Tsn1 cultivars. Preliminary analysis of the B. sorokiniana isolates in Australian wheat cultivars showed that isolates with ToxA19, ToxA25, or ToxA-deficient displayed various degrees of virulence, with the most aggressive isolates observed for those producing ToxA. Differences in spot blotch disease severity between Tsn1 and tsn1 cultivars were observed; however, this was not limited to the ToxA-producing isolates. The overall results suggest that the virulence of the Australian B. sorokiniana isolates is diverse, with the significance of ToxA-Tsn1 interactions depending on individual isolates.

Combined pangenomics and transcriptomics reveals core and redundant virulence processes in a rapidly evolving fungal plant pathogen.

BACKGROUND: Studying genomic variation in rapidly evolving pathogens potentially enables identification of genes supporting their "core biology", being present, functional and expressed by all strains or "flexible biology", varying between strains. Genes supporting flexible biology may be considered to be "accessory", whilst the "core" gene set is likely to be important for common features of a pathogen species biology, including virulence on all host genotypes. The wheat-pathogenic fungus Zymoseptoria tritici represents one of the most rapidly evolving threats to global food security and was the focus of this study. RESULTS: We constructed a pangenome of 18 European field isolates, with 12 also subjected to RNAseq transcription profiling during infection. Combining this data, we predicted a "core" gene set comprising 9807 sequences which were (1) present in all isolates, (2) lacking inactivating polymorphisms and (3) expressed by all isolates. A large accessory genome, consisting of 45% of the total genes, was also defined. We classified genetic and genomic polymorphism at both chromosomal and individual gene scales. Proteins required for essential functions including virulence had lower-than average sequence variability amongst core genes. Both core and accessory genomes encoded many small, secreted candidate effector proteins that likely interact with plant immunity. Viral vector-mediated transient in planta overexpression of 88 candidates failed to identify any which induced leaf necrosis characteristic of disease. However, functional complementation of a non-pathogenic deletion mutant lacking five core genes demonstrated that full virulence was restored by re-introduction of the single gene exhibiting least sequence polymorphism and highest expression. CONCLUSIONS: These data support the combined use of pangenomics and transcriptomics for defining genes which represent core, and potentially exploitable, weaknesses in rapidly evolving pathogens.

The Necrotrophic Pathogen Parastagonospora nodorum Is a Master Manipulator of Wheat Defense.

Parastagonospora nodorum is a necrotrophic pathogen of wheat that is particularly destructive in major wheat-growing regions of the United States, northern Europe, Australia, and South America. P. nodorum secretes necrotrophic effectors that target wheat susceptibility genes to induce programmed cell death (PCD), resulting in increased colonization of host tissue and, ultimately, sporulation to complete its pathogenic life cycle. Intensive research over the last two decades has led to the functional characterization of five proteinaceous necrotrophic effectors, SnTox1, SnToxA, SnTox267, SnTox3, and SnTox5, and three wheat susceptibility genes, Tsn1, Snn1, and Snn3D-1. Functional characterization has revealed that these effectors, in addition to inducing PCD, have additional roles in pathogenesis, including chitin binding that results in protection from wheat chitinases, blocking defense response signaling, and facilitating plant colonization. There are still large gaps in our understanding of how this necrotrophic pathogen is successfully manipulating wheat defense to complete its life cycle. This review summarizes our current knowledge, identifies knowledge gaps, and provides a summary of well-developed tools and resources currently available to study the P. nodorum-wheat interaction, which has become a model for necrotrophic specialist interactions. Further functional characterization of the effectors involved in this interaction and work toward a complete understanding of how P. nodorum manipulates wheat defense will provide fundamental knowledge about this and other necrotrophic interactions. Additionally, a broader understanding of this interaction will contribute to the successful management of Septoria nodorum blotch disease on wheat. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome-wide association mapping of resistance to the foliar diseases septoria nodorum blotch and tan spot in a global winter wheat collection.

Septoria nodorum blotch (SNB) and tan spot, caused by the necrotrophic fungal pathogens Parastagonospora nodorum and Pyrenophora tritici-repentis, respectively, often occur together as a leaf spotting disease complex on wheat (Triticum aestivum L.). Both pathogens produce necrotrophic effectors (NEs) that contribute to the development of disease. Here, genome-wide association analysis of a diverse panel of 264 winter wheat lines revealed novel loci on chromosomes 5A and 5B associated with sensitivity to the NEs SnTox3 and SnTox5 in addition to the known sensitivity genes for NEs Ptr/SnToxA, SnTox1, SnTox3, and SnTox5. Sensitivity loci for SnTox267 and Ptr ToxB were not detected. Evaluation of the panel with five P. nodorum isolates for SNB development indicated the Snn3-SnTox3 and Tsn1-SnToxA interactions played significant roles in disease development along with additional QTL on chromosomes 2A and 2D, which may correspond to the Snn7-SnTox267 interaction. For tan spot, the Tsc1-Ptr ToxC interaction was associated with disease caused by two isolates, and a novel QTL on chromosome 7D was associated with a third isolate. The Tsn1-ToxA interaction was associated with SNB but not tan spot. Therefore some, but not all, of the previously characterized host gene-NE interactions in these pathosystems play significant roles in disease development in winter wheat. Based on these results, breeders should prioritize the selection of resistance alleles at the Tsc1, Tsn1, Snn3, and Snn7 loci as well as the 2A and 7D QTL to obtain good levels of resistance to SNB and tan spot in winter wheat. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01400-5.

A protein kinase-major sperm protein gene hijacked by a necrotrophic fungal pathogen triggers disease susceptibility in wheat.

Septoria nodorum blotch (SNB), a disease caused by the necrotrophic fungal pathogen Parastagonospora nodorum, is a threat to wheat (Triticum aestivum) production worldwide. Multiple inverse gene-for-gene interactions involving the recognition of necrotrophic effectors (NEs) by wheat sensitivity genes play major roles in causing SNB. One interaction involves the wheat gene Snn3 and the P. nodorum NE SnTox3. Here, we used a map-based strategy to clone the Snn3-D1 gene from Aegilops tauschii, the D-genome progenitor of common wheat. Snn3-D1 contained protein kinase and major sperm protein domains, both of which were essential for function as confirmed by mutagenesis. As opposed to other characterized interactions in this pathosystem, a compatible Snn3-D1-SnTox3 interaction was light-independent, and Snn3-D1 transcriptional expression was downregulated by light and upregulated by darkness. Snn3-D1 likely emerged in Ae. tauschii due to an approximately 218-kb insertion that occurred along the west bank of the Caspian Sea. The identification of this new class of NE sensitivity genes combined with the previously cloned sensitivity genes demonstrates that P. nodorum can take advantage of diverse host targets to trigger SNB susceptibility in wheat.

A triple threat: the Parastagonospora nodorum SnTox267 effector exploits three distinct host genetic factors to cause disease in wheat.

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.

The Parastagonospora nodorum necrotrophic effector SnTox5 targets the wheat gene Snn5 and facilitates entry into the leaf mesophyll.

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.

Identification of a Novel ToxA Haplotype of Pyrenophora tritici-repentis from Japan.

Pyrenophora tritici-repentis was described first as a pathogen of wheat (tan spot) in Japan in the 1920s, but, since then, no reports on P. tritici-repentis race structure or its effectors in Japan have been published. In this study, 10 single-spore isolates of P. tritici-repentis were collected from bread wheat in Japan. These isolates were evaluated for virulence on four differential wheat genotypes and tested for the presence/absence of the effector-encoding genes, ToxA and ToxB, in multiplex PCR assays. These isolates were identified as ToxA producers, of which eight were designated as race 2 (ToxA producers) and two were classified as race 1 (ToxA and ToxC producers) based on their virulence patterns. Sequence analysis of the ToxA amplicons from these 10 isolates indicated the presence of a novel ToxA haplotype (denoted PtrA2). A comparative sequence analysis and resequencing of ToxA from reference P. tritici-repentis isolates showed that all previously published ToxA haplotypes in P. tritici-repentis were identical, and are hence denoted PtrA1 in this study. A total of 163 PtrToxA sequences from global origins were already deposited in GenBank and were confirmed identical to PtrA1. Sequence variation in PtrA1 and PtrA2 open reading frames were found at three positions: one synonymous mutation at position 412 (C/G) and two nonsynonymous mutations at positions 342 and 362 that alter amino acid sequence. These mutations did not seem to affect the necrosis development on a ToxA-sensitive wheat genotype when rated for symptoms 5 to 7 days after inoculation. This is the first report correctly confirming the presence of an additional novel ToxA haplotype in P. tritici-repentis for which we have predicted its isoform and updated the ToxA haplotype evolutionary network.

ToxA-Tsn1 Interaction for Spot Blotch Susceptibility in Indian Wheat: An Example of Inverse Gene-for-Gene Relationship.

The ToxA-Tsn1 system is an example of an inverse gene-for-gene relationship. The gene ToxA encodes a host-selective toxin (HST) which functions as a necrotrophic effector and is often responsible for the virulence of the pathogen. The genomes of several fungal pathogens (e.g., Pyrenophora tritici-repentis, Parastagonospora nodorum, and Bipolaris sorokiniana) have been shown to carry the ToxA gene. Tsn1 is a sensitivity gene in the host, whose presence generally helps a ToxA-positive pathogen to cause spot blotch in wheat. Cultivars lacking Tsn1 are generally resistant to spot blotch; this resistance is attributed to a number of other known genes which impart resistance in the absence of Tsn1. In the present study, 110 isolates of B. sorokiniana strains, collected from the ME5A and ME4C megaenvironments of India, were screened for the presence of the ToxA gene; 77 (70%) were found to be ToxA positive. Similarly, 220 Indian wheat cultivars were screened for the presence of the Tsn1 gene; 81 (36.8%) were found to be Tsn1 positive. When 20 wheat cultivars (11 with Tsn1 and 9 with tsn1) were inoculated with ToxA-positive isolates, seedlings of only those carrying the Tsn1 allele (not tsn1) developed necrotic spots surrounded by a chlorotic halo. No such distinction between Tsn1 and tsn1 carriers was observed when adult plants were inoculated. This study suggests that the absence of Tsn1 facilitated resistance against spot blotch of wheat. Therefore, the selection of wheat genotypes for the absence of the Tsn1 allele can improve resistance to spot blotch.

Differential effector gene expression underpins epistasis in a plant fungal disease.

Fungal effector-host sensitivity gene interactions play a key role in determining the outcome of septoria nodorum blotch disease (SNB) caused by Parastagonospora nodorum on wheat. The pathosystem is complex and mediated by interaction of multiple fungal necrotrophic effector-host sensitivity gene systems. Three effector sensitivity gene systems are well characterized in this pathosystem; SnToxA-Tsn1, SnTox1-Snn1 and SnTox3-Snn3. We tested a wheat mapping population that segregated for Snn1 and Snn3 with SN15, an aggressive P. nodorum isolate that produces SnToxA, SnTox1 and SnTox3, to study the inheritance of sensitivity to SnTox1 and SnTox3 and disease susceptibility. Interval quantitative trait locus (QTL) mapping showed that the SnTox1-Snn1 interaction was paramount in SNB development on both seedlings and adult plants. No effect of the SnTox3-Snn3 interaction was observed under SN15 infection. The SnTox3-Snn3 interaction was however, detected in a strain of SN15 in which SnTox1 had been deleted (tox1-6). Gene expression analysis indicates increased SnTox3 expression in tox1-6 compared with SN15. This indicates that the failure to detect the SnTox3-Snn3 interaction in SN15 is due - at least in part - to suppressed expression of SnTox3 mediated by SnTox1. Furthermore, infection of the mapping population with a strain deleted in SnToxA, SnTox1 and SnTox3 (toxa13) unmasked a significant SNB QTL on 2DS where the SnTox2 effector sensitivity gene, Snn2, is located. This QTL was not observed in SN15 and tox1-6 infections and thus suggesting that SnToxA and/or SnTox3 were epistatic. Additional QTLs responding to SNB and effectors sensitivity were detected on 2AS1 and 3AL.

SnTox1, a Parastagonospora nodorum necrotrophic effector, is a dual-function protein that facilitates infection while protecting from wheat-produced chitinases.

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.

Evaluation of a Pyrenophora teres f. teres mapping population reveals multiple independent interactions with a region of barley chromosome 6H.

The necrotrophic fungal pathogen Pyrenophora teres f. teres causes the foliar disease net form net blotch (NFNB) on barley. To investigate the genetics of virulence in the barley- P. teres f. teres pathosystem, we evaluated 118 progeny derived from a cross between the California isolates 15A and 6A on the barley lines Rika and Kombar, chosen based on their differential reactions to isolates 15A and 6A for NFNB disease. Genetic maps generated with SNP, SSR, and AFLP markers were scanned for quantitative trait loci (QTL) associated with virulence in P. teres f. teres. Loci underlying two major QTL, VR1 and VR2, were associated with virulence on Rika barley, accounting for 35% and 20% of the disease reaction type variation, respectively. Two different loci, VK1 and VK2, were shown to underlie two major QTL associated with virulence on Kombar barley accounting for 26% and 19% of the disease reaction type variation, respectively. Progeny isolates harboring VK1, VK2, or VR2 alone were inoculated onto a Rika×Kombar recombinant inbred line mapping population and the susceptibility induced by each pathogen genotype corresponded to the same region on barley chromosome 6H as that identified for the parental isolates 15A and 6A. The data presented here indicate that the P. teres f. teres - barley interaction can at least partially be explained by pathogen-produced necrotrophic effectors (NEs) that interact with dominant barley susceptibility genes resulting in NE triggered susceptibility (NETS).

Comparative genomics of host-specialized populations of Corynespora cassiicola causing target spot epidemics in the southeastern United States.

Numerous plant-pathogenic fungi secrete necrotrophic effectors (syn. host-selective toxins) that are important determinants of pathogenicity and virulence in species that have a necrotrophic lifestyle. Corynespora cassiicola is a necrotrophic fungus causing emerging target spot epidemics in the southeastern United States (US). Previous studies revealed that populations of C. cassiicola from cotton, soybean, and tomato are clonal, host specialized and genetically distinct. Additionally, cassiicolin - the necrotrophic effector identified in some C. cassiicola isolates - is an important toxin for virulence on rubber. It is encoded by seven Cas gene variants. Our goal was to conduct comparative genomic analyses to identify variation among putative necrotrophic effector genes and to determine if lack of one of the mating-types explained clonal populations in C. cassiicola causing outbreaks in the southeastern US and the apparent absence of sexual reproduction worldwide. A total of 12 C. cassiicola genomes, with four each from isolates from tomato, soybean, and cotton, were sequenced using an Illumina Next Seq platform. Each genome was assembled de novo, compared with the reference genome from rubber, and searched for known Cas, and other gene clusters with homologs of secondary metabolites. Cas2 and/or Cas6 were present in isolates from soybean in the southeastern US, whereas Cas1 and Cas2 were present in isolates from cotton in the southeastern US. In addition, several toxin genes, including the T-toxin biosynthetic genes were present in all C. cassiicola from cotton, soybean, and tomato. The mating-type locus was identified in all of the sequenced genomes, with the MAT1-1 idiomorph present in all cotton isolates and the rubber isolate, whereas the MAT1-2 idiomorph was present in all soybean isolates. We developed a PCR-based marker for mating-type in C. cassiicola. Both mating types were present in isolates from tomato. Thus, C. cassiicola has both mating-types necessary for sexual reproduction, but the absence of both mating-types within soybean and cotton populations could explain clonality in these populations. Variation in necrotrophic effectors may underlie host specialization and disease emergence of target spot on cotton, soybean, and tomato in the southeastern US.

Optimization of highly efficient exogenous-DNA-free Cas9-ribonucleoprotein mediated gene editing in disease susceptibility loci in wheat (Triticum aestivum L.).

The advancement of precision engineering for crop trait improvement is important in the face of rapid population growth, climate change, and disease. To this end, targeted double-stranded break technology using RNA-guided Cas9 has been adopted widely for genome editing in plants. Agrobacterium or particle bombardment-based delivery of plasmids encoding Cas9 and guide RNA (gRNA) is common, but requires optimization of expression and often results in random integration of plasmid DNA into the plant genome. Recent advances have described gene editing by the delivery of Cas9 and gRNA as pre-assembled ribonucleoproteins (RNPs) into various plant tissues, but with moderate efficiency in resulting regenerated plants. In this report we describe significant improvements to Cas9-RNP mediated gene editing in wheat. We demonstrate that Cas9-RNP assays in protoplasts are a fast and effective tool for rational selection of optimal gRNAs for gene editing in regenerable immature embryos (IEs), and that high temperature treatment enhances gene editing rates in both tissue types. We also show that Cas9-mediated editing persists for at least 14 days in gold particle bombarded wheat IEs. The regenerated edited wheat plants in this work are recovered at high rates in the absence of exogenous DNA and selection. With this method, we produce knockouts of a set of three homoeologous genes and two pathogenic effector susceptibility genes, engineering insensitivity to corresponding necrotrophic effectors produced by Parastagonospora nodorum. The establishment of highly efficient, exogenous DNA-free gene editing technology holds promise for accelerated trait diversity production in an expansive array of crops.

Characterization of Effector-Target Interactions in Necrotrophic Pathosystems Reveals Trends and Variation in Host Manipulation.

Great strides have been made in defining the details of the plant defense response involving biotrophic fungal and bacterial pathogens. The groundwork for the current model was laid by H.H. Flor and others who defined the gene-for-gene hypothesis, which is now known to involve effector-triggered immunity (ETI). PAMP-triggered immunity (PTI) is also a highly effective response to most pathogens because of the recognition of common pathogen molecules by pattern recognition receptors. In this article, we consider the three pathogens that make up the foliar disease complex of wheat, Zymoseptoria tritici, Pyrenophora tritici-repentis, and Parastagonospora nodorum, to review the means by which necrotrophic pathogens circumvent, or outright hijack, the ETI and PTI pathways to cause disease.

Role of Effector-Sensitivity Gene Interactions and Durability of Quantitative Resistance to Septoria Nodorum Blotch in Eastern U.S. Wheat.

Important advances have been made in understanding the relationship of necrotrophic effectors (NE) and host sensitivity (Snn) genes in the Parastagonospora nodorum-wheat pathosystem. Yet much remains to be learned about the role of these interactions in determining wheat resistance levels in the field, and there is mixed evidence on whether breeding programs have selected against Snn genes due to their role in conferring susceptibility. SNB occurs ubiquitously in the U.S. Atlantic seaboard, and the environment is especially well suited to field studies of resistance to natural P. nodorum populations, as there are no other important wheat leaf blights. Insights into the nature of SNB resistance have been gleaned from multi-year data on phenotypes and markers in cultivars representative of the region's germplasm. In this perspective article, we review the evidence that in this eastern region of the U.S., wheat cultivars have durable quantitative SNB resistance and Snn-NE interactions are of limited importance. This conclusion is discussed in light of the relevant available information from other parts of the world.