Plant growth-promoting rhizobacteria (PGPR) are beneficial microorganisms that colonize the rhizosphere of many plant species and confer beneficial effects, such as an increase in plant growth. PGPR are also well known as inducers of systemic resistance to pathogens in plants. However, the molecular mechanisms involved locally after direct perception of these bacteria by plant cells still remain largely unknown. Burkholderia phytofirmans strain PsJN is an endophytic PGPR that colonizes grapevine and protects the plant against the grey mould disease caused by Botrytis cinerea. This report focuses on local defence events induced by B. phytofirmans PsJN after perception by the grapevine cells. It is demonstrated that, after addition to cell suspension cultures, the bacteria were tightly attaching to plant cells in a way similar to the grapevine non-host bacteria Pseudomonas syringae pv. pisi. B. phytofirmans PsJN perception led to a transient and monophasic extracellular alkalinization but no accumulation of reactive oxygen species or cell death were detected. By contrast, challenge with P. syringae pv. pisi induced a sustained and biphasic extracellular alkalinization, a two phases oxidative burst, and a HR-like response. Perception of the PGPR also led to the production of salicylic acid (SA) and the expression of a battery of defence genes that was, however, weaker in intensity compared with defence gene expression triggered by the non-host bacteria. Some defence genes up-regulated after B. phytofirmans PsJN challenge are specifically induced by exogenous treatment with SA or jasmonic acid, suggesting that both signalling pathways are activated by the PGPR in grapevine.
In this study, we have constructed and expressed inverted repeat chimeras from the first exons of the six known hydrophobins of the fungus Cladosporium fulvum, the causal agent of tomato leaf mold. We used quantitative PCR to measure specifically the expression levels of the hydrophobins. The targeted genes are silenced to different degrees, but we also detected clear changes in the expression levels of nontargeted genes. This work highlights the difficulties that are likely to be encountered when attempting to silence more than one gene in a multigene family.Most filamentous fungi produce hydrophobins, which are small moderately hydrophobic proteins that are characterized by a strictly conserved array of eight cysteines (36). Two distinct types have been identified: class I and class II hydrophobins. These proteins have been extensively reviewed in recent years and play important roles in the formation of aerial hyphae, spore production and dispersal, the stabilization of fruiting body structures, and the virulence of some pathogenic fungi (3,12,26,27,28,29,31,32,(35)(36)(37)(38). In many cases, individual fungal species have more than one hydrophobin gene. For example, the tomato pathogen Cladosporium fulvum has at least six hydrophobins: HCf-1, HCf-2, HCf-3, and HCf-4 are typical class I hydrophobins, and HCf-5 and HCf-6 are class II hydrophobins.Targeted gene deletion of HCf-1 demonstrated that HCf-1 is a major determinant of conidial hydrophobicity and, thus, contributes to the ability of conidia to be dispersed by water (34). While no evident phenotype was observed in mutants lacking HCf-2, HCf-3, HCf-4, and HCf-6, we were unable to isolate a mutant lacking HCf-5. In the presence of a multigene family, it is possible that different hydrophobins complement each other. On the other hand, the inability to isolate a knockout is often considered to be an indication that a gene is essential for viability.As an alternative to allelic replacement, RNA-mediated interference (RNAi) has emerged as an effective method for silencing gene expression (2,15,19,30). Specific inhibition of gene expression by RNAi has been demonstrated in a range of organisms from an initial report on Caenorhabditis elegans (5), trypanosomes (39), Drosophila melanogaster (10), mammalian cells (4), and the yeast Cryptococcus neoformans (14). In fungi, RNAi has been demonstrated in Neurospora crassa, where inverted repeat transgenes forming hairpin RNAs are efficient activators of RNA silencing (7). Since RNA-dependent silencing occurs in C. fulvum (8), we decided to use this tool to analyze the function of the six hydrophobins by simultaneously silencing up to six hydrophobin genes in C. fulvum. In this paper, we report success in obtaining targeted gene silencing of selected hydrophobins, including the simultaneous silencing of all hydrophobins in individual transformants. However, we also highlight the unexpected effects on targeted and nontargeted genes, including gene overexpression, and discuss the limits when applying this technique.Sil...
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