Jafargholi Imani

The root endophytic fungus Piriformospora indica requires host cell death for proliferation during mutualistic symbiosis with barley.

Fungi of the recently defined order Sebacinales (Basidiomycota) are involved in a wide spectrum of mutualistic symbioses (including mycorrhizae) with various plants, thereby exhibiting a unique potential for biocontrol strategies. The axenically cultivable root endophyte Piriformospora indica is a model organism of this fungal order. It is able to increase biomass and grain yield of crop plants. In barley, the endophyte induces local and systemic resistance to fungal diseases and to abiotic stress. To elucidate the lifestyle of P. indica, we analyzed its symbiotic interaction and endophytic development in barley roots. We found that fungal colonization increases with root tissue maturation. The root tip meristem showed no colonization, and the elongation zone showed mainly intercellular colonization. In contrast, the differentiation zone was heavily infested by inter- and intracellular hyphae and intracellular chlamydospores. The majority of hyphae were present in dead rhizodermal and cortical cells that became completely filled with chlamydospores. In some cases, hyphae penetrated cells and built a meshwork around plasmolyzed protoplasts, suggesting that the fungus either actively kills cells or senses cells undergoing endogenous programmed cell death. Seven days after inoculation, expression of barley BAX inhibitor-1 (HvBI-1), a gene capable of inhibiting plant cell death, was attenuated. Consistently, fungal proliferation was strongly inhibited in transgenic barley overexpressing GFP-tagged HvBI-1, which shows that P. indica requires host cell death for proliferation in differentiated barley roots. We suggest that the endophyte interferes with the host cell death program to form a mutualistic interaction with plants.

Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase–encoding genes confers strong resistance to Fusarium species

Significance We demonstrate that host-induced gene silencing (HIGS) targeting the fungal sterol 14α-demethylase (CYP51) genes restricts Fusarium infection in plants. Fusarium diseases have a significant impact not only on global grain production, but also on food safety because of grain contamination with mycotoxins. We capitalized on the knowledge that demethylation inhibitor fungicides target cytochrome P450 lanosterol C-14α-demethylase. In Fusarium graminearum (Fg), this enzyme is encoded by three paralogous genes. Transgenic Arabidopsis and barley expressing a double-stranded RNA targeting all three CYP51 genes exhibited complete immunity to Fg. Our results provide proof-of-concept that HIGS of the CYP51 genes is an effective strategy for controlling Fusarium, demonstrating that HIGS is a powerful tool, which could revolutionize crop plant protection. Head blight, which is caused by mycotoxin-producing fungi of the genus Fusarium, is an economically important crop disease. We assessed the potential of host-induced gene silencing targeting the fungal cytochrome P450 lanosterol C-14α-demethylase (CYP51) genes, which are essential for ergosterol biosynthesis, to restrict fungal infection. In axenic cultures of Fusarium graminearum, in vitro feeding of CYP3RNA, a 791-nt double-stranded (ds)RNA complementary to CYP51A, CYP51B, and CYP51C, resulted in growth inhibition [half-maximum growth inhibition (IC50) = 1.2 nM] as well as altered fungal morphology, similar to that observed after treatment with the azole fungicide tebuconazole, for which the CYP51 enzyme is a target. Expression of the same dsRNA in Arabidopsis and barley rendered susceptible plants highly resistant to fungal infection. Microscopic analysis revealed that mycelium formation on CYP3RNA-expressing leaves was restricted to the inoculation sites, and that inoculated barley caryopses were virtually free of fungal hyphae. This inhibition of fungal growth correlated with in planta production of siRNAs corresponding to the targeted CYP51 sequences, as well as highly efficient silencing of the fungal CYP51 genes. The high efficiency of fungal inhibition suggests that host-induced gene-silencing targeting of the CYP51 genes is an alternative to chemical treatments for the control of devastating fungal diseases.

Detection and identification of bacteria intimately associated with fungi of the order Sebacinales

Because of their beneficial impact on plants, the highly diverse mycorrhizal fungi grouped in the order Sebacinales lay claim to high ecological and agricultural significance. Here, we describe for the first time associations of Sebacinoid members with bacteria. Using quantitative PCR, denaturating gradient gel electrophoresis and fluorescence in situ hybridization, we detected an intimate association between Piriformospora indica and Rhizobium radiobacter, an α‐Proteobacterium. The stability of the association, vertical transmission of the bacteria during asexual fungal reproduction and fungal plant colonization was monitored using R. radiobacter‐specific primers. Treatment of mycelium or fungal protoplasts with antibiotics highly efficient against the free bacteria failed to cure the fungus. Barley seedlings dip‐inoculated with R. radiobacter showed growth promotion and systemic resistance to the powdery mildew fungus Blumeria graminis comparable to P. indica inoculation. By screening additional isolates of the Sebacina vermifera complex, three species‐specific associations with bacteria from the genera Paenibacillus, Acinetobacter and Rhodococcus were found. These findings suggest that Sebacinales species regularly undergo complex interactions involving host plants and bacteria reminiscent of other ectomycorrhizal and endomycorrhizal associations.

Transcriptome and metabolome profiling of field-grown transgenic barley lack induced differences but show cultivar-specific variances

The aim of the present study was to assess possible adverse effects of transgene expression in leaves of field-grown barley relative to the influence of genetic background and the effect of plant interaction with arbuscular mycorrhizal fungi. We conducted transcript profiling, metabolome profiling, and metabolic fingerprinting of wild-type accessions and barley transgenics with seed-specific expression of (1,3-1, 4)-β-glucanase (GluB) in Baronesse (B) as well as of transgenics in Golden Promise (GP) background with ubiquitous expression of codon-optimized Trichoderma harzianum endochitinase (ChGP). We found more than 1,600 differential transcripts between varieties GP and B, with defense genes being strongly overrepresented in B, indicating a divergent response to subclinical pathogen challenge in the field. In contrast, no statistically significant differences between ChGP and GP could be detected based on transcriptome or metabolome analysis, although 22 genes and 4 metabolites were differentially abundant when comparing GluB and B, leading to the distinction of these two genotypes in principle component analysis. The coregulation of most of these genes in GluB and GP, as well as simple sequence repeat-marker analysis, suggests that the distinctive alleles in GluB are inherited from GP. Thus, the effect of the two investigated transgenes on the global transcript profile is substantially lower than the effect of a minor number of alleles that differ as a consequence of crop breeding. Exposing roots to the spores of the mycorrhizal Glomus sp. had little effect on the leaf transcriptome, but central leaf metabolism was consistently altered in all genotypes.

Over-expression of the cell death regulator BAX inhibitor-1 in barley confers reduced or enhanced susceptibility to distinct fungal pathogens

BAX inhibitor-1 (BI-1) is a conserved cell death regulator protein that inhibits mammalian BAX-induced cell death in yeast, animals and plants. Additionally, HvBI-1 suppresses defense responses and resistance to the powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) when over-expressed in single epidermal cells of barley. To test the potential of ectopic expression of BI-1 to influence fungal interactions with crop plants, we produced stable transgenic barley plants expressing a green fluorescing protein (GFP) fusion of HvBI-1 under control of the cauliflower mosaic virus 35S promoter. GFP-HvBI-1 plants were fertile and did not display obvious developmental alterations when compared to wild type parents. GFP-HvBI-1 plants were more resistant to single cell death induced by ballistic delivery of a mammalian proapototic BAX expression construct and more susceptible to biotrophic Bgh. Microscopic observation of the interaction phenotype revealed that enhanced susceptibility, i.e. a higher degree of successful establishment of haustoria in epidermal cells, was associated with a reduced frequency of hypersensitive cell death reactions. In contrast, young seedlings of GFP-HvBI-1 barley were more resistant to Fusarium graminearum than wild type or azygous controls. Hence the effect of GFP-HvBI-1 on the outcome of a particular plant–fungus interaction appeared dependent on the lifestyle of the pathogen.

Insect peptide metchnikowin confers on barley a selective capacity for resistance to fungal ascomycetes pathogens.

The potential of metchnikowin, a 26-amino acid residue proline-rich antimicrobial peptide synthesized in the fat body of Drosophila melanogaster was explored to engineer disease resistance in barley against devastating fungal plant pathogens. The synthetic peptide caused strong in vitro growth inhibition (IC50 value ∼1 μM) of the pathogenic fungus Fusarium graminearum. Transgenic barley expressing the metchnikowin gene in its 52-amino acid pre-pro-peptide form under the control of the inducible mannopine synthase (mas) gene promoter from the Ti plasmid of Agrobacterium tumefaciens displayed enhanced resistance to powdery mildew as well as Fusarium head blight and root rot. In response to these pathogens, metchnikowin accumulated in plant apoplastic space, specifying that the insect signal peptide is functional in monocotyledons. In vitro and in vivo tests revealed that the peptide is markedly effective against fungal pathogens of the phylum Ascomycota but, clearly, less active against Basidiomycota fungi. Importantly, germination of the mutualistic basidiomycete mycorrhizal fungus Piriformospora indica was affected only at concentrations beyond 50 μM. These results suggest that antifungal peptides from insects are a valuable source for crop plant improvements and their differential activities toward different phyla of fungi denote a capacity for insect peptides to be used as selective measures on specific plant diseases.

Transgenic expression of gallerimycin, a novel antifungal insect defensin from the greater wax moth Galleria mellonella, confers resistance to pathogenic fungi in tobacco.

Abstract A cDNA encoding gallerimycin, a novel antifungal peptide from the greater wax moth Galleria mellonella, was isolated from a cDNA library of genes expressed during innate immune response in the caterpillars. Upon ectopic expression of gallerimycin in tobacco, using Agrobacterium tumefaciens as a vector, gallerimycin conferred resistance to the fungal pathogens Erysiphe cichoracearum and Sclerotinia minor. Quantification of gallerimycin mRNA in transgenic tobacco by real-time PCR confirmed transgenic expression under control of the inducible mannopine synthase promoter. Leaf sap and intercellular washing fluid from transgenic tobacco inhibited in vitro germination and growth of the fungal pathogens, demonstrating that gallerimycin is secreted into intercellular spaces. The feasibility of the use of gallerimycin to counteract fungal diseases in crop plants is discussed.

Ectopic Expression of Constitutively Activated RACB in Barley Enhances Susceptibility to Powdery Mildew and Abiotic Stress

Small RAC/ROP-family G proteins regulate development and stress responses in plants. Transient overexpression and RNA interference experiments suggested that the barley (Hordeum vulgare) RAC/ROP protein RACB is involved in susceptibility to the powdery mildew fungus Blumeria graminis f. sp. hordei. We created transgenic barley plants expressing the constitutively activated RACB mutant racb-G15V under control of the maize (Zea mays) ubiquitin 1 promoter. Individuals of the T1 generation expressing racb-G15V were significantly more susceptible to B. graminis when compared to segregating individuals that did not express racb-G15V. Additionally, racb-G15V-expressing plants showed delayed shoot development from the third leaf stage on, downward rolled leaves, and stunted roots. Expression of racb-G15V decreased photosynthetic CO2-assimilation rates and transpiration of nonstressed leaves. In contrast, racb-G15V-expressing barley leaves, when detached from water supply, showed increased water loss and enhanced transpiration. Water loss was associated with reduced responsiveness to abscisic acid in regard to transpiration when compared to segregants not expressing racb-G15V. Hence, RACB might be a common signaling element in response to both biotic and abiotic stress.

The importance of soil microbial activity for the supply of iron to sorghum and rape

Abstract It is generally accepted that soil microorganisms play an important role in producing siderophores which enhance the availability of soil Fe to higher plants. There is not much direct experimental evidence to support this supposition, however, because it is difficult to grow plants under sterile conditions over long periods. The object of this investigation was to test whether a sterile soil medium impairs Fe translocation from the soil to plant roots. The plant species selected are of agronomical importance, namely rape (Brassica napus L.) and sorghum (Sorghum bicolor L.). The latter a graminaceous species which is able to excrete phytosiderophores from the roots into the soil which allows Fe to be mobilized and transported to plant roots. Sorghum and rape were grown for 18 and 21 days, respectively, in a non-sterile soil (control) and in the same soil which was sterilized before plant cultivation. In a further treatment, the sterile grown rape plants were supplied with Fe EDDHA 1 week before harvest in order to test whether a poor growth of plants grown in the sterile soil was caused by an insufficient Fe supply. Plants cultivated on the sterile soil were significantly retarded in root and shoot growth. This was especially true for rape which produced very small leaves. Plants responded immediately to the Fe addition which induced a vigorous growth. This clearly shows that the poor growth in the sterile soil was caused at least in part by an insufficient Fe supply. In neither plant species was yellowing of young leaves observed as a symptom typical of insufficient Fe supply. From this follows that retardation of plant growth is a more sensitive indicator of an insufficient Fe supply than is yellowing of young leaves at least for rape and sorghum. This finding is of agronomical importance since a reduced growth because of insufficient Fe supply is much more difficult to identify as Fe deficieny than Fe chlorosis (yellow leaves). Iron concentrations in roots and leaves of the sterile grown plants were significantly lower than the corresponding concentrations in the non-sterile grown plants. From these findings it can be concluded that soil microbial activity is essential for Fe acquisition by soil-grown rape. Similarily, sorghum which is able to release siderophores from the roots, requires soil microbial activity to ensure satisfactory Fe supply.

Expression of barley BAX Inhibitor‐1 in carrots confers resistance to Botrytis cinerea

SUMMARY BAX Inhibitor-1 (BI-1) is a protein that controls heterologous BAX-induced cell death, the hypersensitive reaction and abiotic stress-induced cell death in plants. When over-expressed in epidermal cells of barley, barley BI-1 induces susceptibility to the biotrophic fungal pathogen Blumeria graminis. When we expressed barley BI-1 in carrot susceptible to the necrotrophic fungus Botrytis cinerea, we obtained BI-1-mediated resistance to fungus-induced leaf cell death and less fungal spreading on the leaves. Barley BI-1 also mediated resistance to Chalara elegans in carrot roots. The results support the idea that cell death inhibition is an applicable approach to control cell-death-inducing pathogens in crop plants.