During seed development, plants detect the paternal genome dose through gene expression from the paternally derived genome in the endosperm. To achieve this, the homologous genes in the maternally derived genome are silenced by polycomb repressive complex 2 (PRC2), and loss of PRC2 function or excess paternal genome dose both lead to seed abortion through the so-called 'triploid block'. PRC2 requires the activity of accessory proteins of the VERNALIZATION INSENSITIVE 3-LIKE (VEL) family, of which the atypical VEL3 protein is required for endosperm development. Here, we characterize the function of the remaining family members, using CRISPR-Cas9 to generate multiple mutants in Arabidopsis. Loss-of-function analyses establish VEL3 as the sole regulator of primary seed dormancy depth, whereas VEL1 and VRN5 act redundantly alongside VEL3 to prevent seed abortion but without affecting dormancy. VEL2 and VIN3 exhibited no detectable influence on seed abortion or dormancy. Comparative RNA-seq of mature endosperm revealed that both vel3-1 and vel1-2 vrn5-9 mutants upregulate programmed cell-death genes, whereas vel3-1 uniquely deregulates chromatin remodellers and metabolic repressors, consistent with heterochromatin relaxation at germination loci and elevated metabolic activity. Our results establish that the role of VEL3 in seed dormancy is independent of PRC2, as they suggest the involvement of novel members of central cell and endosperm PRC2 complexes in regulating the maternally derived genome in the endosperm.

Mother plants play an important role in the control of dormancy and dispersal characters of their progeny. In Arabidopsis seed dormancy is imposed by the embryo-surrounding tissues of the endosperm and seed coat. Here we show that VERNALIZATION5/VIN3-LIKE 3 (VEL3) maintains maternal control over progeny seed dormancy by establishing an epigenetic state in the central cell that primes the depth of primary seed dormancy later established during seed maturation. VEL3 colocalises with MSI1 in the nucleolus and associates with a histone deacetylase complex. Furthermore, VEL3 preferentially associates with pericentromeric chromatin and is required for deacetylation and H3K27me3 deposition established in the central cell. The epigenetic state established by maternal VEL3 is retained in mature seeds, and controls seed dormancy in part through repression of programmed cell death-associated gene ORE1. Our data demonstrates a mechanism by which maternal control of progeny seed physiology persists post-shedding, maintaining parental control of seed behaviour.

Mother plants play an important role in the control of dormancy and dispersal characters of their progeny. In Arabidopsis seed dormancy is imposed by the embryo-surrounding tissues of the endosperm and seed coat. Here we show that the VERNALIZATION5/VIN3-LIKE 3 (VEL3) gene maintains maternal control over progeny seed dormancy by establishing an epigenetic state early in endosperm development that primes the depth of primary seed dormancy later established during seed maturation. VEL3 relocates MSI1 to the nucleolus and associates with other components of the histone deacetylase complex (HDAC), and is required for gene repression via PRC2 and HDAC at pericentromeric regions established in the central cell. Interestingly, the epigenetic state established by the maternal VEL3 is retained trans-generationally in mature seeds, and controls seed dormancy through the repression of programmed cell death-associated gene ORE1 . Our data demonstrates a novel mechanism by which maternal control of progeny seed physiology persists post-shedding, maintaining parental control of seed behaviour.

Agricultural soil harbors a diverse microbiome that can form beneficial relationships with plants, including the inhibition of plant pathogens. Pseudomonas spp. are one of the most abundant bacterial genera in the soil and rhizosphere and play important roles in promoting plant health. However, the genetic determinants of this beneficial activity are only partially understood. Here, we genetically and phenotypically characterize the Pseudomonas fluorescens population in a commercial potato field, where we identify strong correlations between specialized metabolite biosynthesis and antagonism of the potato pathogens Streptomyces scabies and Phytophthora infestans . Genetic and chemical analyses identified hydrogen cyanide and cyclic lipopeptides as key specialized metabolites associated with S. scabies inhibition, which was supported by in planta biocontrol experiments. We show that a single potato field contains a hugely diverse and dynamic population of Pseudomonas bacteria, whose capacity to produce specialized metabolites is shaped both by plant colonization and defined environmental inputs.

Linking of wheat and barley orthologue candidates. (XLSX 2951Â kb)

BACKGROUND:Nonhost resistance (NHR) protects plants against a vast number of non-adapted pathogens which implicates a potential exploitation as source for novel disease resistance strategies. Aiming at a fundamental understanding of NHR a global analysis of transcriptome reprogramming in the economically important Triticeae cereals wheat and barley, comparing host and nonhost interactions in three major fungal pathosystems responsible for powdery mildew (Blumeria graminis ff. ssp.), cereal blast (Magnaporthe sp.) and leaf rust (Puccinia sp.) diseases, was performed. RESULTS:In each pathosystem a significant transcriptome reprogramming by adapted- or non-adapted pathogen isolates was observed, with considerable overlap between Blumeria, Magnaporthe and Puccinia. Small subsets of these general pathogen-regulated genes were identified as differentially regulated between host and corresponding nonhost interactions, indicating a fine-tuning of the general pathogen response during the course of co-evolution. Additionally, the host- or nonhost-related responses were rather specific for each pair of adapted and non-adapted isolates, indicating that the nonhost resistance-related responses were to a great extent pathosystem-specific. This pathosystem-specific reprogramming may reflect different resistance mechanisms operating against non-adapted pathogens with different lifestyles, or equally, different co-option of the hosts by the adapted isolates to create an optimal environment for infection. To compare the transcriptional reprogramming between wheat and barley, putative orthologues were identified. Within the wheat and barley general pathogen-regulated genes, temporal expression profiles of orthologues looked similar, indicating conserved general responses in Triticeae against fungal attack. However, the comparison of orthologues differentially expressed between host and nonhost interactions revealed fewer commonalities between wheat and barley, but rather suggested different host or nonhost responses in the two cereal species. CONCLUSIONS:Taken together, our results suggest independent co-evolutionary forces acting on host pathosystems mirrored by barley- or wheat-specific nonhost responses. As a result of evolutionary processes, at least for the pathosystems investigated, NHR appears to rely on rather specific plant responses.

Eighty‐eight Australian and 10 international barley cultivars were assessed for resistance to the barley stripe (yellow) rust pathogen, Puccinia striiformis f. sp. hordei (Psh). All cultivars were tested for seedling resistance to two UK‐derived isolates of Psh (11.01 and 83.39) that were shown to differ in virulence based on responses on 16 differential barley genotypes. The 98 barley cultivars differed substantially in stripe rust response; 45% were susceptible to Psh 11.01, 53% to Psh 83.39 and 44% to both isolates. The observed diverse infection types (ITs) suggest the presence of both known and uncharacterized resistance. However, further multipathotype tests are required for accurate gene postulation. The Yerong × Franklin (Y×F) doubled haploid (DH) population was phenotypically assessed as seedlings using both Psh isolates. Yerong and Franklin were immune and highly resistant, respectively, to both isolates used in this study. Marker‐trait and QTL mapping identified a major effect on the long arm of chromosome 7H contributed by Franklin in response to all isolates. The resistance of Yerong was mapped to 113·96 and 169·38 cM on chromosome 5HL in response to Psh 11.01 and 83.39, respectively. The Psh resistance sources identified in this study can be used for further genetic analysis and introgression for varietal improvement.

Pattern recognition receptors (PRRs) belonging to the multigene family of receptor-like kinases (RLKs) are the sensing devices of plants for microbe- or pathogen-associated molecular patterns released from microbial organisms. Here we describe Rnr8 (for Required for non-host resistance 8) encoding HvLEMK1, a LRR-malectin domain-containing transmembrane RLK that mediates non-host resistance of barley to the non-adapted wheat powdery mildew fungus Blumeria graminis f.sp. tritici. Transgenic barley lines with silenced HvLEMK1 allow entry and colony growth of the non-adapted pathogen, although sporulation was reduced and final colony size did not reach that of the adapted barley powdery mildew fungus B. graminis f.sp. hordei. Transient expression of the barley or wheat LEMK1 genes enhanced resistance in wheat to the adapted wheat powdery mildew fungus while expression of the same genes did not protect barley from attack by the barley powdery mildew fungus. The results suggest that HvLEMK1 is a factor mediating non-host resistance in barley and quantitative host resistance in wheat to the wheat powdery mildew fungus.

Perception of pathogen (or microbe)-associated molecular patterns (PAMPs/MAMPs) by pattern recognition receptors (PRRs) is a key component of plant innate immunity. The Arabidopsis PRR EF-Tu receptor (EFR) recognizes the bacterial PAMP elongation factor Tu (EF-Tu) and its derived peptide elf18. Previous work revealed that transgenic expression of AtEFR in Solanaceae confers elf18 responsiveness and broad-spectrum bacterial disease resistance. In this study, we developed a set of bioassays to study the activation of PAMP-triggered immunity (PTI) in wheat. We generated transgenic wheat (Triticum aestivum) plants expressing AtEFR driven by the constitutive rice actin promoter and tested their response to elf18. We show that transgenic expression of AtEFR in wheat confers recognition of elf18, as measured by the induction of immune marker genes and callose deposition. When challenged with the cereal bacterial pathogen Pseudomonas syringae pv. oryzae, transgenic EFR wheat lines had reduced lesion size and bacterial multiplication. These results demonstrate that AtEFR can be transferred successfully from dicot to monocot species, further revealing that immune signalling pathways are conserved across these distant phyla. As novel PRRs are identified, their transfer between plant families represents a useful strategy for enhancing resistance to pathogens in crops.

Leaf rust, caused by the foliar pathogen Puccinia triticina is a major disease of wheat in the southern region of Brazil and invariably impacts on production, being responsible for high yield losses. The Brazilian wheat cultivar Toropi has proven, durable adult plant resistance (APR) to leaf rust, which uniquely shows a pre-haustorial resistance phenotype. In this study we aimed to understand the interaction between P. triticina and the pre-haustorial APR in Toropi by quantitatively evaluating the temporal transcription profiles of selected genes known to be related to infection and defense in wheat. The expression profiles of 15 selected genes varied over time, grouping into six expression profile groups. The expression profiles indicated the induction of classical defence pathways in response to pathogen development, but also the potential modification of Toropi's cellular status for the benefit of the pathogen. Classical defence genes, including peroxidases, β-1,3-glucanases and an endochitinase were expressed both early (pre-haustorial) and late (post-haustorial) over the 72 h infection time course, while induction of transcription of other infection-related genes with a potential role in defence, although variable was maintained through-out. These genes directly or indirectly had a role in plant lignification, oxidative stress, the regulation of energy supply, water and lipid transport, and cell cycle regulation. The early induction of transcription of defence-related genes supports the pre-haustorial resistance phenotype in Toropi, providing a valuable source of genes controlling leaf rust resistance for wheat breeding.

Barley stripe mosaic virus (BSMV) has emerged as a vector for virus-induced gene silencing (VIGS) in cereals, having been used to study a number of genes involved in resistance in both wheat and barley. However, the effects of the BSMV vector on plant physiology and disease resistance in plants remains unexplored. The BSMV inoculation control vector, BSMV:GFP was shown to cause severe viral symptoms in wheat, displaying chlorosis, leaf curling and growth inhibition typical of the symptoms seen in BSMV-infected barley. These viral symptoms were accompanied by induction of genes implicated in defense against pathogens, namely PR1, PR4, PR5, PR10 and PAL. Subsequent inoculation of BSMV:GFP-infected wheat with a wheat pathotype of Magnaporthe oryzae, the blast pathogen, resulted in decreased susceptibility. Penetration of epidermal cells and subsequent multiple cell colonization by M. oryzae was significantly reduced. This increased restriction of pathogen growth observed for BSMV:GFP infections with and without the viral coat protein gene. However, prior infection with BSMV:GFP had no effect on the development of a compatible isolate of Blumeria graminis f. sp. tritici, the causal agent of powdery mildew.

Arabidopsis thaliana is known to produce the phytoalexin camalexin in response to abiotic and biotic stress. Here we studied the mechanisms of tolerance to camalexin in the fungus Botrytis cinerea, a necrotrophic pathogen of A. thaliana. Exposure of B. cinerea to camalexin induces expression of BcatrB, an ABC transporter that functions in the efflux of fungitoxic compounds. B. cinerea inoculated on wild-type A. thaliana plants yields smaller lesions than on camalexin-deficient A. thaliana mutants. A B. cinerea strain lacking functional BcatrB is more sensitive to camalexin in vitro and less virulent on wild-type plants, but is still fully virulent on camalexin-deficient mutants. Pre-treatment of A. thaliana with UV-C leads to increased camalexin accumulation and substantial resistance to B. cinerea. UV-C-induced resistance was not seen in the camalexin-deficient mutants cyp79B2/B3, cyp71A13, pad3 or pad2, and was strongly reduced in ups1. Here we demonstrate that an ABC transporter is a virulence factor that increases tolerance of the pathogen towards a phytoalexin, and the complete restoration of virulence on host plants lacking this phytoalexin.

Recent findings are consistent with a slow but constant shift towards reduced sensitivity of Mycosphaerella graminicola to azole fungicides, which target the CYP51 gene. The goal of this study was to elucidate the evolutionary mechanisms through which CYP51-based mutations associated with altered sensitivity have evolved in M. graminicola over space and time. To accomplish this, we sequenced and compared a portion of the CYP51 gene encompassing the main mutations associated with altered sensitivity towards demethylation inhibitor fungicides. The CYP51 gene showed an extraordinary dynamic shift consistent with a selective haplotype replacement both in space and in time. No mutations associated with increased resistance to azoles were found in non-European populations. These mutations were also absent in the oldest collections from Europe, whereas they dominated in the recent European populations. Intragenic recombination was identified as an important evolutionary process in populations affected by high fungicide selection, suggesting the creation of novel alleles among existing mutations as a potential source of novel resistance alleles. We propose that CYP51 mutations giving resistance in M. graminicola arose only locally (perhaps in Denmark or the UK) and were then spread eastward across Europe through wind-dispersed ascospores. We conclude that recurring cycles of recombination coupled with selection due to the widespread use of azole fungicides will increase the frequency of novel mutants or recombinants with higher resistance. Long-distance gene flow due to wind dispersal of ascospores will move the resulting new alleles to new areas following the prevailing wind directions. A selective replacement favouring haplotypes with various coding mutations at the target site for azole fungicides during the last 5-10 years is the most likely cause of the decrease in sensitivity reported for many azole fungicides in the same period.

SUMMARY Sterol demethylation inhibitors (DMIs) represent one of the largest groups of systemic fungicides that have been used to control agriculturally important fungal pathogens. Knowledge regarding the evolution of fungicide resistance in agricultural ecosystems is fragmentary and a better understanding of the processes driving the development of DMI resistance in populations of fungal pathogens is needed by plant pathologists and the agrochemical industry. We considered some of these processes using approaches based on molecular population and quantitative genetics. Five Mycosphaerella graminicola populations sampled from unsprayed wheat fields on four continents were assayed for eight restriction fragment length polymorphism (RFLP) markers and their level of tolerance to cyproconazole. DMI fungicides such as cyproconazole inhibit the enzyme eburicol 14-alpha-demethylase. The gene encoding this target, CYP51, was sequenced for all isolates. We found unimodal, continuous variations in cyproconazole tolerance among the M. graminicola isolates sampled from individual fields, consistent with a polygenic mode of inheritance. We also found that population differentiation for cyproconazole tolerance (Q(ST)) among the five M. graminicola populations was significantly higher than the corresponding population differentiation for neutral RFLP markers (G(ST)), suggesting that selection for cyproconazole tolerance in the Swiss population has already led to local adaptation that can be seen even in an unsprayed population. The Swiss population displayed the highest level of tolerance to cyproconazole, in addition to a lower than expected quantitative variation in fungicide tolerance and a skewed distribution, indicating that selection had increased the overall tolerance of this population. Further analysis with DNA sequencing showed that the population from Switzerland was dominated by isolates with several point mutations and a 6-bp deletion in CYP51. This deletion and one of the point mutations were previously related to increased resistance in field isolates. The fungal population from Oregon sampled from an unsprayed resistant host cultivar displayed the same gene diversity in RFLP loci but higher cyproconazole tolerance and quantitative variation in tolerance than the fungal population from the same field sampled from an unsprayed susceptible host cultivar.

The grapevine (Vitis) secondary metabolite resveratrol is considered a phytoalexin, which protects the plant from Botrytis cinerea infection. Laccase activity displayed by the fungus is assumed to detoxify resveratrol and to facilitate colonization of grape. We initiated a functional molecular genetic analysis of B. cinerea laccases by characterizing laccase genes and evaluating the phenotype of targeted gene replacement mutants. Two different laccase genes from B. cinerea were characterized, Bclcc1 and Bclcc2. Only Bclcc2 was strongly expressed in liquid cultures in the presence of either resveratrol or tannins. This suggested that Bclcc2, but not Bclcc1, plays an active role in the oxidation of both resveratrol and tannins. Gene replacement mutants in the Bclcc1 and Bclcc2 gene were made to perform a functional analysis. Only Bclcc2 replacement mutants were incapable of converting both resveratrol and tannins. When grown on resveratrol, both the wild type and the Bclcc1 replacement mutant showed inhibited growth, whereas Bclcc2 replacement mutants were unaffected. Thus, contrary to the current theory, BcLCC2 does not detoxify resveratrol but, rather, converts it into compounds that are more toxic for the fungus itself. The Bclcc2 gene was expressed during infection of B. cinerea on a resveratrol-producing host plant, but Bclcc2 replacement mutants were as virulent as the wild-type strain on various hosts. The activation of a plant secondary metabolite by a pathogen introduces a new dimension to plant-pathogen interactions and the phytoalexin concept.