The antibiotic peptaibol alamethicin from Trichoderma permeabilises Arabidopsis root apical meristem and epidermis but is antagonised by cellulase-induced resistance to alamethicin.

BACKGROUND: Trichoderma fungi live in the soil rhizosphere and are beneficial for plant growth and pathogen resistance. Several species and strains are currently used worldwide in co-cultivation with crops as a biocontrol alternative to chemical pesticides even though little is known about the exact mechanisms of the beneficial interaction. We earlier found alamethicin, a peptide antibiotic secreted by Trichoderma, to efficiently permeabilise cultured tobacco cells. However, pre-treatment with Trichoderma cellulase made the cells resistant to subsequent alamethicin, suggesting a potential mechanism for plant tolerance to Trichoderma, needed for mutualistic symbiosis. RESULTS: We here investigated intact sterile-grown Arabidopsis thaliana seedlings germinated in water or growth medium. These could be permeabilised by alamethicin but not if pretreated with cellulase. By following the fluorescence from the membrane-impermeable DNA-binding probe propidium iodide, we found alamethicin to mainly permeabilise root tips, especially the apical meristem and epidermis cells, but not the root cap and basal meristem cells nor cortex cells. Alamethicin permeabilisation and cellulase-induced resistance were confirmed by developing a quantitative in situ assay based on NADP-isocitrate dehydrogenase accessibility. The combined assays also showed that hyperosmotic treatment after the cellulase pretreatment abolished the induced cellulase resistance. CONCLUSION: We here conclude the presence of cell-specific alamethicin permeabilisation, and cellulase-induced resistance to it, in root tip apical meristem and epidermis of the model organism A. thaliana. We suggest that contact between the plasma membrane and the cell wall is needed for the resistance to remain. Our results indicate a potential mode for the plant to avoid negative effects of alamethicin on plant growth and localises the point of potential damage and response. The results also open up for identification of plant genetic components essential for beneficial effects from Trichoderma on plants.

Host cell ploidy underlying the fungal feeding site is a determinant of powdery mildew growth and reproduction.

Golovinomyces orontii is an obligate biotrophic powdery mildew (PM) that colonizes Arabidopsis thaliana and agronomic species. It establishes a specialized feeding structure in epidermal cells to fuel its extensive surface hyphal growth and reproduction. Previously, endoreduplication was identified in Arabidopsis mesophyll cells underlying the fungal feeding site, presumably to meet the metabolic demands imposed by the fungus. Furthermore, the cell cycle transcription factor MYB3R4 was shown to regulate this process. Herein, PM-induced endoreduplication is further characterized and three additional factors influencing host ploidy in cells underlying the fungal feeding site are identified. While mutations in PUX2 and PMR6 reduce basal ploidy, mutations in PMR5 (and MYB3R4) abrogate the PM-induced ploidy increase. Moreover, analysis of pmr5 microarray data suggests that PMR5 acts upstream of a MYB3R transcription factor such as MYB3R4 to control PM-induced ploidy. Induced endoreduplication occurs exclusively in mesophyll cells underlying the fungal feeding site at 5 days postinoculation, concomitant with PM reproduction. Gene copy number increases and chromatin remains decondensed, suggesting active, elevated gene expression. Cell ploidy underlying the fungal feeding site is highly correlated with the extent of PM growth and reproduction for these mutants, indicating that (induced) mesophyll cell ploidy is a PM susceptibility determinant.

Local DNA hypomethylation activates genes in rice endosperm.

Cytosine methylation silences transposable elements in plants, vertebrates, and fungi but also regulates gene expression. Plant methylation is catalyzed by three families of enzymes, each with a preferred sequence context: CG, CHG (H = A, C, or T), and CHH, with CHH methylation targeted by the RNAi pathway. Arabidopsis thaliana endosperm, a placenta-like tissue that nourishes the embryo, is globally hypomethylated in the CG context while retaining high non-CG methylation. Global methylation dynamics in seeds of cereal crops that provide the bulk of human nutrition remain unknown. Here, we show that rice endosperm DNA is hypomethylated in all sequence contexts. Non-CG methylation is reduced evenly across the genome, whereas CG hypomethylation is localized. CHH methylation of small transposable elements is increased in embryos, suggesting that endosperm demethylation enhances transposon silencing. Genes preferentially expressed in endosperm, including those coding for major storage proteins and starch synthesizing enzymes, are frequently hypomethylated in endosperm, indicating that DNA methylation is a crucial regulator of rice endosperm biogenesis. Our data show that genome-wide reshaping of seed DNA methylation is conserved among angiosperms and has a profound effect on gene expression in cereal crops.

Substrate and Plant Genotype Strongly Influence the Growth and Gene Expression Response to Trichoderma afroharzianum T22 in Sugar Beet.

Many strains of Trichoderma fungi have beneficial effects on plant growth and pathogen control, but little is known about the importance of plant genotype, nor the underlying mechanisms. We aimed to determine the effect of sugar beet genotypic variation on Trichoderma biostimulation. The effect of Trichoderma afroharzianum T22 on sugar beet inbred genotypes were investigated in soil and on sterile agar medium regarding plant growth, and by quantitative reverse transcriptase-linked polymerase chain reaction (qRT-PCR) analysis for gene expression. In soil, T22 application induced up to 30% increase or decrease in biomass, depending on plant genotype. In contrast, T22 treatment of sterile-grown seedlings resulted in a general decrease in fresh weight and root length across all sugar beet genotypes. Root colonization of T22 did not vary between the sugar beet genotypes. Sand- and sterile-grown roots were investigated by qRT-PCR for expression of marker genes for pathogen response pathways. Genotype-dependent effects of T22 on, especially, the jasmonic acid/ethylene expression marker PR3 were observed, and the effects were further dependent on the growth system used. Thus, both growth substrate and sugar beet genotype strongly affect the outcome of inoculation with T. afroharzianum T22.

New clothes for the jasmonic acid receptor COI1: delayed abscission, meristem arrest and apical dominance.

In a screen for delayed floral organ abscission in Arabidopsis, we have identified a novel mutant of CORONATINE INSENSITIVE 1 (COI1), the F-box protein that has been shown to be the jasmonic acid (JA) co-receptor. While JA has been shown to have an important role in senescence, root development, pollen dehiscence and defense responses, there has been little focus on its critical role in floral organ abscission. Abscission, or the detachment of organs from the main body of a plant, is an essential process during plant development and a unique type of cell separation regulated by endogenous and exogenous signals. Previous studies have indicated that auxin and ethylene are major plant hormones regulating abscission; and here we show that regulation of floral organ abscission is also controlled by jasmonic acid in Arabidopsis thaliana. Our characterization of coi1-1 and a novel allele (coi1-37) has also revealed an essential role in apical dominance and floral meristem arrest. In this study we provide genetic evidence indicating that delayed abscission 4 (dab4-1) is allelic to coi1-1 and that meristem arrest and apical dominance appear to be evolutionarily divergent functions for COI1 that are governed in an ecotype-dependent manner. Further characterizations of ethylene and JA responses of dab4-1/coi1-37 also provide new information suggesting separate pathways for ethylene and JA that control both floral organ abscission and hypocotyl growth in young seedlings. Our study opens the door revealing new roles for JA and its interaction with other hormones during plant development.