To detect molecules with elicitor properties from Pythium oligandrum, cell wall protein fractions (CWPs) were extracted from 10 P. oligandrum isolates and examined for elicitor activity in sugar beet and wheat. P. oligandrum isolates were divided into two groups based on the number of major proteins in CWP: isolates with two major proteins (D-type) and isolates with one major protein (S-type). Sugar beet seedlings treated with both types of CWP through their roots showed enhanced activities of phenylalanine ammonia lyase and chitinase, and D-type-treated seedlings also showed significantly higher cell wall-bound phenolic compounds, mainly ferulic acid, compared with the distilled-water-treatment control. Damping-off severity was significantly reduced on seedlings treated with both types of CWP compared with the control, following challenge with Rhizoctonia solani AG2-2. Both types of CWP significantly reduced the number of infected spikelets developed from the injected spikelet compared with the control, following challenge with Fusarium graminearum. Neither type of CWP resulted in any reduction in pathogen growth rate in plate tests. These results demonstrate that CWPs of P. oligandrum have elicitor properties in sugar beet and wheat.

When the biocontrol agent Pythium oligandrum (PO) colonizes the rhizosphere, it suppresses bacterial wilt disease in tomato ( Solanum lycopersicum cv. Micro-Tom) caused by Ralstonia solanacearum , and a homogenate of its mycelia exhibits elicitor activity, inducing an ethylene (ET)-dependent defence response in Micro-Tom. Since salicylic acid (SA) and jasmonic acid (JA) play an important role in plant defence responses to pathogens, the involvement of SA-and JAdependent signal transduction pathways in resistance to R. solanacearum was investigated in tomato roots treated with a mycelial homogenate of PO. Bacterial wilt disease was also suppressed in tomato cv. Moneymaker treated with the PO homogenate. However, the SA-inducible PR-1 ( P6 ) gene was not up-regulated in either Micro-Tom or Moneymaker. SA did not accumulate in homogenate-treated roots in comparison with distilled water-treated controls, even 24 h after inoculation. Induced resistance against R. solanacearum was not compromised in SA-non-accumulating NahG transgenic plants treated with the PO homogenate. On the other hand, the expression of the JA-responsive gene for the basic PR-6 protein was induced in both tomato cultivars treated with the PO homogenate. Furthermore, quantitative disease assays showed that the induced resistance against R. solanacearum was compromized in PO homogenate-treated jai1-1 mutant plants defective in JA signalling. These results indicated that the JA-dependent signalling pathway is required for PO-induced resistance against R. solanacearum in tomato.

Pythium oligandrum (PO) is a mycoparasite on a wide range of fungi and suppresses diseases caused by fungal pathogens when colonizing the rhizosphere. PO and its cell wall proteins (CWPs) have elicitor activity that induces defence responses in plants. The potential of a mycelial homogenate of PO to suppress bacterial diseases was studied in roots of tomato ( Lycopersicon esculentum cv. Micro-Tom) inoculated with Ralstonia solanacearum . PO-treated plants showed enhanced resistance to R. solanacearum and reduction in severity of wilt symptoms. As ethylene often acts as one of the signal molecules for induced resistance, its production following treatment of tomato roots with the mycelial homogenate or CWP of PO was measured. The level of ethylene in PO-and CWP-treated plants was transiently elevated six-to 11-fold at 4-8 h after treatment, followed by high expression of three basic ethylene-inducible defence-related genes ( PR-2b , PR-3b and PR-5b ). Analysis of PR-5b gene expression in the leaves of PO-and CWP-treated plants suggested that PR gene expression was induced systemically. The expression of LeERF2 and LeETR4 , which confer an ethylenedependent signalling pathway, was also significantly accelerated by such treatments. These results indicate that PO has the potential to control bacterial wilt disease and that CWP may play an important role in the induction of resistance to R. solanacearum accompanying the activation of the ethylene-dependent signalling pathway.

The expression of LeATL6, an ortholog of Arabidopsis ATL6 that encodes a RING-H2 finger protein, was induced in tomato roots treated with a cell wall protein fraction (CWP) elicitor of the biocontrol agent Pythium oligandrum. The LeATL6 protein was expressed as a fusion protein with a maltose-binding protein (MBP) in Escherichia coli, and it catalyzed the transfer of ubiquitin to the MBP moiety on incubation with ubiquitin, the ubiquitin-activating enzyme E1, and the ubiquitin-conjugating enzyme E2; this indicated that LeATL6 represents ubiquitin ligase E3. LeATL6 expression also was induced by elicitor treatment of jail-1 mutant tomato cells in which the jasmonic acid (JA)-mediated signaling pathway was impaired; however, JA-dependent expression of the basic PR-6 and TPI-1 genes that encode proteinase inhibitor II and I, respectively, was not induced in elicitor-treated jail-1 mutants. Furthermore, transient overexpression of LeATL6 under the control of the Cauliflower mosaic virus 35S promoter induced the basic PR6 and TPI-1 expression in wild tomato but not in the jail-1 mutant. In contrast, LeATL6 overexpression did not activate salicylic acid-responsive acidic PR-1 and PR-2 promoters in wild tomato. These results indicated that elicitor-responsive LeATL6 probably regulates JA-dependent basic PR6 and TPI-1 gene expression in tomato. The LeATL6-associated ubiquitin/proteasome system may contribute to elicitor-activated defense responses via a JA-dependent signaling pathway in plants.

SUMMARY We previously reported that cell wall protein fractions (CWPs) of the biocontrol agent Pythium oligandrum have elicitor properties in sugar beet and wheat. Here we have examined the effect of treatment with the D-type of CWP, a fraction that contains two major forms (POD-1 and POD-2), on the induction of defence-related genes in sugar beet. Using PCR-based cDNA library subtraction, we identified five genes that were highly expressed in response to CWP treatment. The five genes are probably of oxalate oxidase-like germin (OxOLG), glutathione S-transferase (GST), 5-enol-pyruvylshikimate-phosphate synthase (EPSPS), phenylalanine ammonia-lyase (PAL) and aspartate aminotransferase (AAT). In addition, we purified and characterized POD-1 and POD-2 and found that POD-1 induced all five genes, whereas POD-2 induced three of the genes, but not OxOLG or GST. A sugar beet bioassay indicated that CWP, POD-1 and POD-2 are each sufficient to induce resistance to sugar beet seedling disease caused by Aphanomyces cochlioides. Although carbohydrate analyses indicated that POD proteins were glycoproteins with similar carbohydrate compositions, containing approximately 15.0% carbohydrate by weight, their peptide portions have elicitor activity. Furthermore, cDNAs of POD-1 and POD-2 proteins were cloned, and the deduced amino acid sequences were found to be 82.9% identical. Characterization of their molecular structures indicated that they have an elicitin domain followed by a C-terminal domain with a high frequency of Ser, Thr, Ala and Pro, which is structurally similar to class III elicitins. However, phylogenetic analysis with 22 representative elicitin and elicitin-like proteins showed that POD-1 and POD-2 are distinct from previously defined elicitin and elicitin-like proteins. Therefore, POD-1 and POD-2 are novel oomycete cell wall elicitin-like glycoproteins.