Fusarium Head Blight Modifies Fungal Endophytic Communities During Infection of Wheat Spikes.

Fusarium head blight (FHB) is a devastating disease of wheat heads. It is caused by several species from the genus Fusarium. Several endophytic fungi also colonize wheat spikes asymptomatically. Pathogenic and commensal fungi share and compete for the same niche and thereby influence plant performance. Understanding the natural dynamics of the fungal community and how the pre-established species react to pathogen attack can provide useful information on the disease biology and the potential use of some of these endophytic organisms in disease control strategies. Fungal community composition was assessed during anthesis as well as during FHB attack in wheat spikes during 2016 and 2017 in two locations. Community metabarcoding revealed that endophyte communities are dominated by basidiomycete yeasts before anthesis and shift towards a more opportunistic ascomycete-rich community during kernel development. These dynamics are interrupted when Fusarium spp. colonize wheat spikes. The Fusarium pathogens appear to exclude other fungi from floral tissues as they are associated with a reduction in community diversity, especially in the kernel which they colonize rapidly. Similarly, the presence of several endophytes was negatively correlated with Fusarium spp. and linked with spikes that stayed healthy despite exposure to the pathogen. These endophytes belonged to the genera Cladosporium, Itersonillia and Holtermanniella. These findings support the hypothesis that some naturally occurring endophytes could outcompete or prevent FHB and represent a source of potential biological control agents in wheat.

A cerato-platanin protein SsCP1 targets plant PR1 and contributes to virulence of Sclerotinia sclerotiorum.

Cerato-platanin proteins (CPs), which are secreted by filamentous fungi, are phytotoxic to host plants, but their functions have not been well defined to date. Here we characterized a CP (SsCP1) from the necrotrophic phytopathogen Sclerotinia sclerotiorum. Sscp1 transcripts accumulated during plant infection, and deletion of Sscp1 significantly reduced virulence. SsCP1 could induce significant cell death when expressed in Nicotiana benthamiana. Using yeast two-hybrid, GST pull-down, co-immunoprecipitation and bimolecular florescence complementation, we found that SsCP1 interacts with PR1 in the apoplast to facilitate infection by S. sclerotiorum. Overexpressing PR1 enhanced resistance to the wild-type strain, but not to the Sscp1 knockout strain of S. sclerotiorum. Sscp1-expressing transgenic plants showed increased concentrations of salicylic acid (SA) and higher levels of resistance to several plant pathogens (namely Botrytis cinerea, Alternaria brassicicola and Golovinomyces orontii). Our results suggest that SsCP1 is important for virulence of S. sclerotiorum and that it can be recognized by plants to trigger plant defense responses. Our results also suggest that the SA signaling pathway is involved in CP-mediated plant defense .

Large-Scale Phenomics Identifies Primary and Fine-Tuning Roles for CRKs in Responses Related to Oxidative Stress.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.

Transcriptomic profiling to identify genes involved in Fusarium mycotoxin Deoxynivalenol and Zearalenone tolerance in the mycoparasitic fungus Clonostachys rosea.

BACKGROUND: Clonostachys rosea strain IK726 is a mycoparasitic fungus capable of controlling mycotoxin-producing Fusarium species, including F. graminearum and F. culmorum, known to produce Zearalenone (ZEA) and Deoxynivalenol (DON). DON is a type B trichothecene known to interfere with protein synthesis in eukaryotes. ZEA is a estrogenic-mimicing mycotoxin that exhibits antifungal growth. C. rosea produces the enzyme zearalenone hydrolase (ZHD101), which degrades ZEA. However, the molecular basis of resistance to DON in C. rosea is not understood. We have exploited a genome-wide transcriptomic approach to identify genes induced by DON and ZEA in order to investigate the molecular basis of mycotoxin resistance C. rosea. RESULTS: We generated DON- and ZEA-induced cDNA libraries based on suppression subtractive hybridization. A total of 443 and 446 sequenced clones (corresponding to 58 and 65 genes) from the DON- and ZEA-induced library, respectively, were analysed. DON-induced transcripts represented genes encoding metabolic enzymes such as cytochrome P450, cytochrome c oxidase and stress response proteins. In contrast, transcripts encoding the ZEA-detoxifying enzyme ZHD101 and those encoding a number of ATP-Binding Cassette (ABC) transporter transcripts were highly frequent in the ZEA-induced library. Subsequent bioinformatics analysis predicted that all transcripts with similarity to ABC transporters could be ascribed to only 2 ABC transporters genes, and phylogenetic analysis of the predicted ABC transporters suggested that they belong to group G (pleiotropic drug transporters) of the fungal ABC transporter gene family. This is the first report suggesting involvement of ABC transporters in ZEA tolerance. Expression patterns of a selected set of DON- and ZEA-induced genes were validated by the use of quantitative RT-PCR after exposure to the toxins. The qRT-PCR results obtained confirm the expression patterns suggested from the EST redundancy data. CONCLUSION: The present study identifies a number of transcripts encoding proteins that are potentially involved in conferring resistance to DON and ZEA in the mycoparasitic fungus C. rosea. Whilst metabolic readjustment is potentially the key to withstanding DON, the fungus produces ZHD101 to detoxify ZEA and ABC transporters to transport ZEA or its degradation products out from the fungal cell.

The barley HvNAC6 transcription factor affects ABA accumulation and promotes basal resistance against powdery mildew.

Barley HvNAC6 is a member of the plant-specific NAC (NAM, ATAF1,2, CUC2) transcription factor family and we have shown previously that it acts as a positive regulator of basal resistance in barley against the biotrophic pathogen Blumeria graminis f. sp. hordei (Bgh). In this study, we use a transgenic approach to constitutively silence HvNAC6 expression, using RNA interference (RNAi), to investigate the in vivo functions of HvNAC6 in basal resistance responses in barley in relation to the phytohormone ABA. The HvNAC6 RNAi plants displayed reduced HvNAC6 transcript levels and were more susceptible to Bgh than wild-type plants. Application of exogenous ABA increased basal resistance against Bgh in wild-type plants, but not in HvNAC6 RNAi plants, suggesting that ABA is a positive regulator of basal resistance which depends on HvNAC6. Silencing of HvNAC6 expression altered the light/dark rhythm of ABA levels which were, however, not influenced by Bgh inoculation. The expression of the two ABA biosynthetic genes HvNCED1 and HvNCED2 was compromised, and transcript levels of the ABA conjugating HvBG7 enzyme were elevated in the HvNAC6 RNAi lines, but this effect was not clearly associated with transgene-mediated resistance. Together, these data support a function of HvNAC6 as a regulator of ABA-mediated defence responses for maintenance of effective basal resistance against Bgh.

Biocontrol Effect of Clonostachys rosea on Fusarium graminearum Infection and Mycotoxin Detoxification in Oat (Avena sativa).

Oat (Avena sativa) is susceptible to Fusarium head blight (FHB). The quality of oat grain is threatened by the accumulation of mycotoxins, particularly the trichothecene deoxynivalenol (DON), which also acts as a virulence factor for the main pathogen Fusarium graminearum. The plant can defend itself, e.g., by DON detoxification by UGT-glycosyltransferases (UTGs) and accumulation of PR-proteins, even though these mechanisms do not deliver effective levels of resistance. We studied the ability of the fungal biocontrol agent (BCA) Clonostachys rosea to reduce FHB and mycotoxin accumulation. Greenhouse trials showed that C. rosea-inoculation of oat spikelets at anthesis 3 days prior to F. graminearum inoculation reduced both the amount of Fusarium DNA (79%) and DON level (80%) in mature oat kernels substantially. DON applied to C. rosea-treated spikelets resulted in higher conversion of DON to DON-3-Glc than in mock treated plants. Moreover, there was a significant enhancement of expression of two oat UGT-glycosyltransferase genes in C. rosea-treated oat. In addition, C. rosea treatment activated expression of genes encoding four PR-proteins and a WRKY23-like transcription factor, suggesting that C. rosea may induce resistance in oat. Thus, C. rosea IK726 has strong potential to be used as a BCA against FHB in oat as it inhibits F. graminearum infection effectively, whilst detoxifying DON mycotoxin rapidly.

Fungal endophytes in plants and their relationship to plant disease.

The enigmatic endophytic fungi are beginning to reveal their secrets. Like pathogens, they can manipulate the host for their own benefit to create their own optimal habitat. Some endophytic manipulations induce resistance or otherwise outcompete pathogens and can thus be exploited for biological control. Like pathogens and other symbionts, endophytes produce effector proteins and other molecules, ranging from specialised metabolites, phytohormones and microRNAs, to manipulate their hosts and other microorganisms they meet. There is a continuum from endophyte to pathogen: some organisms can infest or cause disease in some hosts, but not in others. Molecular genetics approaches coupled with functional characterisation have demonstrated their worth for understanding the biological phenomena underlying endophytic fungal interactions.

Identification and Functional Characterisation of Two Oat UDP-Glucosyltransferases Involved in Deoxynivalenol Detoxification.

Oat is susceptible to several Fusarium species that cause contamination with different trichothecene mycotoxins. The molecular mechanisms behind Fusarium resistance in oat have yet to be elucidated. In the present work, we identified and characterised two oat UDP-glucosyltransferases orthologous to barley HvUGT13248. Overexpression of the latter in wheat had been shown previously to increase resistance to deoxynivalenol (DON) and nivalenol (NIV) and to decrease disease the severity of both Fusarium head blight and Fusarium crown rot. Both oat genes are highly inducible by the application of DON and during infection with Fusarium graminearum. Heterologous expression of these genes in a toxin-sensitive strain of Saccharomyces cerevisiae conferred high levels of resistance to DON, NIV and HT-2 toxins, but not C4-acetylated trichothecenes (T-2, diacetoxyscirpenol). Recombinant enzymes AsUGT1 and AsUGT2 expressed in Escherichia coli rapidly lost activity upon purification, but the treatment of whole cells with the toxin clearly demonstrated the ability to convert DON into DON-3-O-glucoside. The two UGTs could therefore play an important role in counteracting the Fusarium virulence factor DON in oat.

Regulation of Tomato Specialised Metabolism after Establishment of Symbiosis with the Endophytic Fungus Serendipita indica.

Specialised metabolites produced during plant-fungal associations often define how symbiosis between the plant and the fungus proceeds. They also play a role in the establishment of additional interactions between the symbionts and other organisms present in the niche. However, specialised metabolism and its products are sometimes overlooked when studying plant-microbe interactions. This limits our understanding of the specific symbiotic associations and potentially future perspectives of their application in agriculture. In this study, we used the interaction between the root endophyte Serendipita indica and tomato (Solanum lycopersicum) plants to explore how specialised metabolism of the host plant is regulated upon a mutualistic symbiotic association. To do so, tomato seedlings were inoculated with S. indica chlamydospores and subjected to RNAseq analysis. Gene expression of the main tomato specialised metabolism pathways was compared between roots and leaves of endophyte-colonised plants and tissues of endophyte-free plants. S. indica colonisation resulted in a strong transcriptional response in the leaves of colonised plants. Furthermore, the presence of the fungus in plant roots appears to induce expression of genes involved in the biosynthesis of lignin-derived compounds, polyacetylenes, and specific terpenes in both roots and leaves, whereas pathways producing glycoalkaloids and flavonoids were expressed in lower or basal levels.

The Fungal Endophyte Penicillium olsonii ML37 Reduces Fusarium Head Blight by Local Induced Resistance in Wheat Spikes.

The fungal endophyte Penicillium olsonii ML37 is a biocontrol agent of Fusarium head blight in wheat (caused by Fusarium graminearum), which has shown a limited direct inhibition of fungal growth in vitro. We used RNA-seq and LC-MS/MS analyses to elucidate metabolic interactions of the three-way system Penicillium-wheat-Fusarium in greenhouse experiments. We demonstrated that P. olsonii ML37 colonises wheat spikes and transiently activates plant defence mechanisms, as pretreated spikes show a faster and stronger expression of the defence metabolism during the first 24 h after pathogen inoculation. This effect was transient and the expression of the same genes was lower in the pathogen-infected spikes than in those infected by P. olsonii alone. This response to the endophyte includes the transcriptional activation of several WRKY transcription factors. This early activation is associated with a reduction in FHB symptoms and significantly lower levels of the F. graminearum metabolites 15-acetyl-DON and culmorin. An increase in the Penicillium-associated metabolite asperphanamate confirms colonisation by the endophyte. Our results suggest that the mode of action used by P. olsonii ML37 is via a local defence activation in wheat spikes, and that this fungus has potential as a novel biological alternative in wheat disease control.

Disease-reducing effect of Chromolaena odorata extract on sheath blight and other rice diseases.

Sheath blight caused by Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) is a major cause of crop loss in intensive rice production systems. No economically viable control methods have been developed. We screened aqueous extracts of common herbal plants that could reduce sheath blight lesions and found that foliar spraying and seed soaking application of extracts of either fresh or dried leaves of Chromolaena odorata gave up to 68% reduction in sheath blight lesion lengths under controlled and semi-field conditions. The observed reductions were not dependent on growth conditions of C. odorata and rice cultivar. The effect was observed until 21 days after inoculation and was not dependent on microbial activity. Under semi-field conditions, extracts also reduced severity of other important rice diseases, i.e., blast (Pyricularia oryzae) using foliar spray (up to 45%), brown spot (Bipolaris oryzae) using seed treatment (up to 57%), and bacterial blight (Xanthomonas oryzae pv. oryzae) using both application methods (up to 50%).

A Sesquiterpene Synthase from the Endophytic Fungus Serendipita indica Catalyzes Formation of Viridiflorol.

Interactions between plant-associated fungi and their hosts are characterized by a continuous crosstalk of chemical molecules. Specialized metabolites are often produced during these associations and play important roles in the symbiosis between the plant and the fungus, as well as in the establishment of additional interactions between the symbionts and other organisms present in the niche. Serendipita indica, a root endophytic fungus from the phylum Basidiomycota, is able to colonize a wide range of plant species, conferring many benefits to its hosts. The genome of S. indica possesses only few genes predicted to be involved in specialized metabolite biosynthesis, including a putative terpenoid synthase gene (SiTPS). In our experimental setup, SiTPS expression was upregulated when the fungus colonized tomato roots compared to its expression in fungal biomass growing on synthetic medium. Heterologous expression of SiTPS in Escherichia coli showed that the produced protein catalyzes the synthesis of a few sesquiterpenoids, with the alcohol viridiflorol being the main product. To investigate the role of SiTPS in the plant-endophyte interaction, an SiTPS-over-expressing mutant line was created and assessed for its ability to colonize tomato roots. Although overexpression of SiTPS did not lead to improved fungal colonization ability, an in vitro growth-inhibition assay showed that viridiflorol has antifungal properties. Addition of viridiflorol to the culture medium inhibited the germination of spores from a phytopathogenic fungus, indicating that SiTPS and its products could provide S. indica with a competitive advantage over other plant-associated fungi during root colonization.

A novel transcription factor UvCGBP1 regulates development and virulence of rice false smut fungus Ustilaginoidea virens.

Ustilaginoidea virens, causing rice false smut (RFS) is an economically important ascomycetous fungal pathogen distributed in rice-growing regions worldwide. Here, we identified a novel transcription factor UvCGBP1 (Cutinase G-box binding protein) from this fungus, which is unique to ascomycetes. Deletion of UvCGBP1 affected development and virulence of U. virens. A total of 865 downstream target genes of UvCGBP1 was identified using ChIP-seq and the most significant KEGG enriched functional pathway was the MAPK signaling pathway. Approximately 36% of target genes contain the AGGGG (G-box) motif in their promoter. Among the targets, deletion of UvCGBP1 affected transcriptional and translational levels of UvPmk1 and UvSlt2, both of which were important in virulence. ChIP-qPCR, yeast one-hybrid and EMSA confirmed that UvCGBP1 can bind the promoter of UvPmk1 or UvSlt2. Overexpression of UvPmk1 in the ∆UvCGBP1-33 mutant restored partially its virulence and hyphae growth, indicating that UvCGBP1 could function via the MAPK pathway to regulate fungal virulence. Taken together, this study uncovered a novel regulatory mechanism of fungal virulence linking the MAPK pathway mediated by a G-box binding transcription factor, UvCGBP1.

Succession of the fungal endophytic microbiome of wheat is dependent on tissue-specific interactions between host genotype and environment.

Fungi living inside plants affect many aspects of plant health, but little is known about how plant genotype influences the fungal endophytic microbiome. However, a deeper understanding of interactions between plant genotype and biotic and abiotic environment in shaping the plant microbiome is of significance for modern agriculture, with implications for disease management, breeding and the development of biocontrol agents. For this purpose, we analysed the fungal wheat microbiome from seed to plant to seeds and studied how different potential sources of inoculum contributed to shaping of the microbiome. We conducted a large-scale pot experiment with related wheat cultivars over one growth-season in two environments (indoors and outdoors) to disentangle the effects of host genotype, abiotic environment (temperature, humidity, precipitation) and fungi present in the seed stock, air and soil on the succession of the endophytic fungal communities in roots, flag leaves and seeds at harvest. The communities were studied with ITS1 metabarcoding and environmental climate factors were monitored during the experimental period. Host genotype, tissue type and abiotic factors influenced fungal communities significantly. The effect of host genotype was mostly limited to leaves and roots, and was location-independent. While there was a clear effect of plant genotype, the relatedness between cultivars was not reflected in the microbiome. For the phyllosphere microbiome, location-dependent weather conditions factors largely explained differences in abundance, diversity, and presence of genera containing pathogens, whereas the root communities were less affected by abiotic factors. Our findings suggest that airborne fungi are the primary inoculum source for fungal communities in aerial plant parts whereas vertical transmission is likely to be insignificant. In summary, our study demonstrates that host genotype, environment and presence of fungi in the environment shape the endophytic fungal community in wheat over a growing season.

Analysis of early events in the interaction between Fusarium graminearum and the susceptible barley (Hordeum vulgare) cultivar Scarlett.

A proteomic analysis was conducted to map the events during the initial stages of the interaction between the fungal pathogen Fusarium graminearum and the susceptible barley cultivar Scarlett. Quantification of fungal DNA demonstrated a sharp increase in fungal biomass in barley spikelets at 3 days after inoculation. This coincided with the appearance of discrete F. graminearum-induced proteolytic fragments of ß-amylase. Based on these results, analysis of grain proteome changes prior to extensive proteolysis enabled identification of barley proteins responding early to infection by the fungus. In total, the intensity of 51 protein spots was significantly changed in F. graminearum-infected spikelets and all but one were identified. These included pathogenesis-related proteins, proteins involved in energy metabolism, secondary metabolism and protein synthesis. A single fungal protein of unknown function was identified. Quantitative real-time RT-PCR analysis of selected genes showed a correlation between high gene expression and detection of the corresponding proteins. Fungal genes encoding alkaline protease and endothiapepsin were expressed during 1-3 days after inoculation, making them candidates for generation of the observed ß-amylase fragments. These fragments have potential to be developed as proteome-level markers for fungal infection that are also informative about grain protein quality.

Identification and characterization of barley RNA-directed RNA polymerases.

RNA-directed RNA polymerases (RDRs) play crucial roles in the RNA silencing response of plants by enhancing and maintaining silencing signals. At least two members of the RDR group, namely RDR1 and RDR6, are implicated in defence against plant viruses. RDRs have so far only been characterized in dicot species. In this report, we identified and characterized HvRDR1, HvRDR2 and HvRDR6 genes in the monocot plant barley (Hordeum vulgare). We analysed their expression under various biotic and abiotic stresses including fungal and viral infections, salicylic acid treatment as well as during plant development. The different classes and subclasses of barley RDRs displayed contrasting expression patterns during pathogen challenge and development suggesting their involvement in specific regulatory pathways. Their response to heat and salicylic acid treatment suggests a conserved pattern of expression of these genes between monocot and dicot plant species. The existence of two HvRDR1 and two HvRDR6 genes suggests an evolutionary selection for specialization in response to biotic and abiotic stresses after gene duplication.

Insights into the community structure and lifestyle of the fungal root endophytes of tomato by combining amplicon sequencing and isolation approaches with phytohormone profiling.

Little is known about the influence of host genotype and phytohormones on the composition of fungal endophytic communities. We investigated the influence of host genotype and phytohormones on the structure of the fungal endophytic communities of tomato roots by amplicon sequencing of the ITS1 region and combined this approach with isolation and functional characterization of the isolates. A significant effect of the host genotype on the dominant fungal species was found by comparing the cultivars Castlemart and UC82B and, surprisingly, root pathogens were among the most abundant taxa. In contrast, smaller changes in the relative abundance of the dominant species were found in mutants impaired in jasmonic acid biosynthesis (def1) and ethylene biosynthesis (8338) compared to the respective wild types. However, def1 showed significantly higher species richness compared to the wild type. Analysis of the phytohormone profiles of these genotypes indicates that changes in the phytohormone balance may contribute to this difference in species richness. Assessing the lifestyle of isolated fungi on tomato seedlings revealed the presence of both beneficial endophytes and latent pathogens in roots of asymptomatic plants, suggesting that the interactions between members of the microbiome maintain the equilibrium in the community preventing pathogens from causing disease.

A 2-kb Mycovirus Converts a Pathogenic Fungus into a Beneficial Endophyte for Brassica Protection and Yield Enhancement.

Mycoviruses are viruses that infect fungi, and hypovirulence-associated mycoviruses have the potential to control fungal diseases. However, it is unclear how mycovirus-mediated hypovirulent strains live and survive in the field, and no mycovirus has been applied for field crop protection. In this study, we found that a previously identified small DNA mycovirus (SsHADV-1) can convert its host, Sclerotinia sclerotiorum, from a typical necrotrophic pathogen to a beneficial endophytic fungus. SsHADV-1 downregulates the expression of key pathogenicity factor genes in S. sclerotiorum during infection. When growing in rapeseed, the SsHADV-1-infected strain DT-8 significantly regulates the expression of rapeseed genes involved in defense, hormone signaling, and circadian rhythm pathways. As a result, plant growth is promoted and disease resistance is enhanced. Field experiments showed that spraying DT-8 at the early flowering stage can reduce the disease severity of rapeseed stem rot by 67.6% and improve yield by 14.9%. Moreover, we discovered that SsHADV-1 could also infect other S. sclerotiorum strains on DT-8-inoculated plants and that DT-8 could be recovered from dead plants. These findings suggest that the mycoviruses may have the ability to shape the origin of endophytism. Our discoveries suggest that mycoviruses may influence the origin of endophytism and may also offer a novel strategy for disease control in which mycovirus-infected strains are used to improve crop health and release mycoviruses into the field.

Transcriptional regulation by an NAC (NAM-ATAF1,2-CUC2) transcription factor attenuates ABA signalling for efficient basal defence towards Blumeria graminis f. sp. hordei in Arabidopsis.

ATAF1 is a member of a largely uncharacterized plant-specific gene family encoding NAC transcription factors, and is induced in response to various abiotic and biotic stimuli in Arabidopsis thaliana. Previously, we showed that a mutant allele of ATAF1 compromises penetration resistance in Arabidopsis with respect to the non-host biotrophic pathogen Blumeria graminis f. sp. hordei (Bgh). In this study, we have used genome-wide transcript profiling to characterize signalling perturbations in ataf1 plants following Bgh inoculation. Comparative transcriptomic analyses identified an over-representation of abscisic acid (ABA)-responsive genes, including the ABA biosynthesis gene AAO3, which is significantly induced in ataf1 plants compared to wild-type plants following inoculation with Bgh. Additionally, we show that Bgh inoculation results in decreased endogenous ABA levels in an ATAF1-dependent manner, and that the ABA biosynthetic mutant aao3 showed increased penetration resistance to Bgh compared to wild-type plants. Furthermore, we show that ataf1 plants show ABA-hyposensitive phenotypes during seedling development and germination. Our data support a negative correlation between ABA levels and penetration resistance, and identify ATAF1 as a new stimuli-dependent attenuator of ABA signalling for the mediation of efficient penetration resistance in Arabidopsis upon Bgh attack.

Effects of beta-1,3-glucan from Septoria tritici on structural defence responses in wheat.

The accumulation of the pathogenesis-related (PR) proteins beta-1,3-glucanase and chitinase and structural defence responses were studied in leaves of wheat either resistant or susceptible to the hemibiotrophic pathogen Septoria tritici. Resistance was associated with an early accumulation of beta-1,3-glucanase and chitinase transcripts followed by a subsequent reduction in level. Resistance was also associated with high activity of beta-1,3-glucanase, especially in the apoplastic fluid, in accordance with the biotrophic/endophytic lifestyle of the pathogen in the apoplastic spaces, thus showing the highly localized accumulation of defence proteins in the vicinity of the pathogen. Isoform analysis of beta-1,3-glucanase from the apoplastic fluid revealed that resistance was associated with the accumulation of an endo-beta-1,3-glucanase, previously implicated in defence against pathogens, and a protein with identity to ADPG pyrophosphatase (92%) and germin-like proteins (93%), which may be involved in cell wall reinforcement. In accordance with this, glycoproteins like extensin were released into the apoplast and callose accumulated to a greater extent in cell walls, whereas lignin and polyphenolics were not found to correlate with defence. Treatment of a susceptible wheat cultivar with purified beta-1,3-glucan fragments from cell walls of S. tritici gave complete protection against disease and this was accompanied by increased gene expression of beta-1,3-glucanase and the deposition of callose. Collectively, these data indicate that resistance is dependent on a fast, initial recognition of the pathogen, probably due to beta-1,3-glucan in the fungal cell walls, and this results in the accumulation of beta-1,3-glucanase and structural defence responses, which may directly inhibit the pathogen and protect the host against fungal enzymes and toxins.