Mitochondrial-targeting effector RsIA_CtaG/Cox11 in Rhizoctonia solani AG-1 IA has two functions: plant immunity suppression and cell death induction mediated by a rice cytochrome c oxidase subunit.

Rhizoctonia solani AG-1 IA causes a necrotrophic rice disease and is a serious threat to rice production. To date, only a few effectors have been characterized in AG-1 IA. We previously identified RsIA_CtaG/Cox11 and showed that infiltration of the recombinant protein into rice leaves caused disease-like symptoms. In the present study, we further characterized the functionality of RsIA_CtaG/Cox11. RsIA_CtaG/Cox11 is an alternative transcript of cytochrome c oxidase copper chaperone Cox11 that starts from the second AUG codon, but contains a functional secretion signal peptide. RNA interference with RsIA_CtaG/Cox11 reduced the pathogenicity of AG-1 IA towards rice and Nicotiana benthamiana without affecting its fitness or mycelial morphology. Transient expression of the RsIA_CtaG/Cox11-GFP fusion protein demonstrated the localization of RsIA_CtaG/Cox11 to mitochondria. Agro-infiltration of RsIA_CtaG/Cox11 into N. benthamiana leaves inhibited cell death by BAX and INF1. In contrast to rice, agro-infiltration of RsIA_CtaG/Cox11 did not induce cell death in N. benthamiana. However, cell death was observed when it was coinfiltrated with Os_CoxVIIa, which encodes a subunit of cytochrome c oxidase. Os_CoxVIIa appeared to interact with RsIA_CtaG/Cox11. The cell death triggered by coexpression of RsIA_CtaG/Cox11 and Os_CoxVIIa is independent of the leucine-rich repeat receptor kinases BAK1/SOBIR1 and enhanced the susceptibility of N. benthamiana to AG-1 IA. Two of the three evolutionarily conserved cysteine residues at positions 25 and 126 of RsIA_CtaG/Cox11 were essential for its immunosuppressive activity, but not for cell death induction. This report suggests that RsIA_CtaG/Cox11 appears to have a dual role in immunosuppression and cell death induction during pathogenesis.

Analysis of TabZIP15 transcription factor from Trichoderma asperellum ACCC30536 and its function under pathogenic toxin stress.

The TabZIP15 gene encoding a 396 amino acid (aa) polypeptide in the fungus Trichoderma asperellum ACCC30536 was cloned and characterised. The protein includes a basic region motif (NR-x2-QR-x2-R) and has a pillar-like structure. The 25 basic region/leucine zipper transcription factors (TFs) identified in the T. asperellum genome were divided into YAP (14 TFs), ATF2 (5), GCN4 (2), Zip1 (2), BRLZ (1) and u1 (1) subfamilies based on conserved domains. T. asperellum was cultured in minimal media (MM) control, C-Hungry and N-Hungry medium (to simulate nutrient competition and interaction with pathogens, respectively), and differential expression analysis showed that 14 TabZIP genes (including TabZIP15) were significantly altered under both conditions; TabZIP23 responded strongly to N-Hungry media and TabZIP24 responded strongly to C-Hungry media. However, only YAP genes TabZIP15, TabZIP12 and TabZIP2 were significantly upregulated under both conditions, and expression levels of TabZIP15 were highest. T. asperellum was also cultured in the presence of five fungal pathogenic toxins, and RT-qPCR results showed that TabZIP15 was significantly upregulated in four of the five toxin stress conditions (MM + Rhizoctonia solani, MM + Fusarium oxysporum, MM + Alternaria alternata and MM + Cytospora chrysosperma).

A mitogenic lectin from Rhizoctonia bataticola arrests growth, inhibits metastasis, and induces apoptosis in human colon epithelial cancer cells.

The correlation between colorectal cancer (CRC) progression and altered expression of N-glycans can be considered in search for new biomarkers and anticancer agents to control CRC. Earlier N-glycan specific mitogenic lectin from Rhizoctonia bataticola (RBL) has been reported which has growth inhibitory and apoptotic effect on human ovarian and leukemic cells, but mitogenic effect on normal PBMCs revealing its clinical potential. Here, we report the effect of RBL on human colon cancer HT 29, SW480, and SW620 cell growth and its differential binding to human normal colon and cancer tissues. RBL has strong binding to both primary and metastatic colon cancer cells with MFI of 403, 404, and 192, respectively for HT 29, SW480, and SW620 cells. RBL shows dose and time dependent growth inhibitory effect with IC50 of 5, 6.4, and 6.8 µg/mL, respectively for HT 29, SW480, and SW620 cells. RBL inhibited the clonogenicity of colon carcinoma cells. RBL arrests metastatic SW620 cell growth at S phase, increased hypodiploid population by 6.1%, 14.3%, and 23.2%, respectively at 12, 24, and 36 h. Further, RBL induces SW620 cell apoptosis in time dependent manner, showed increased release of ROS and nuclear degradation compared to lectin untreated control. Adhesion, wound healing, invasion, and migration assays demonstrated anti-metastasis effect of RBL in SW620 cells apart from its growth inhibitory effect. Anti angiogenic effect of RBL was demonstrated by CAM assay. All these results show the promising potential of RBL both as diagnostic and therapeutic agent.

Opportunistic invasive fungal pathogen Macrophomina phaseolina prognosis from immunocompromised humans to potential mitogenic RBL with an exceptional and novel antitumor and cytotoxic effect.

With the ever-increasing risk for fungal infections, one can no longer ignore fungi. It is imperative that clinical manifestations "presume fungus" with their epidemiologic and pathogenic features when evaluating a potentially infected patient. In the high-risk patient groups, fungi with intrinsic resistance to antifungal agents already exist, with a tendency to emerge as opportunistic pathogens. One of the smart pathogens is Macrophomina phaseolina, with the potential to disarm plant, animal, and human immunity. The response prophylaxis may vary from antifungal therapy and surgical measures to biochemical (Rhizoctonia bataticola lectin [RBL] with antitumor and cytotoxic nature) and gene therapeutics.

Biological control of potato black scurf by rhizosphere associated bacteria

The present work was carried out to study the potential of plant rhizosphere associated bacteria for the biocontrol of potato black scurf disease caused by Rhizoctonia solani Khun AG-3. A total of twenty-eight bacteria isolated from diseased and healthy potato plants grown in the soil of Naran and Faisalabad, Pakistan were evaluated for their antagonistic potential. Nine bacterial strains were found to be antagonistic in vitro, reduced the fungal growth and caused the lysis of sclerotia of R. solani in dual culture assay as well as in extracellular metabolite efficacy test. The selected antagonistic strains were further tested for the production and efficacy of volatile and diffusible antibiotics, lytic enzymes and siderophores against R. solani. Selected antagonistic bacteria were also characterized for growth promoting attributes i.e., phosphate solubilization, nitrogen fixation and indole acetic acid production. Biocontrol efficacy and percent yield increase by these antagonists was estimated in greenhouse experiment. Statistical analysis showed that two Pseudomonas spp. StT2 and StS3 were the most effective with 65.1 and 73.9 percent biocontrol efficacy, as well as 87.3 and 98.3 percent yield increase, respectively. Potential antagonistic bacterial strain StS3 showed maximum homology to Pseudomonas sp. as determined by 16S rRNA gene sequencing. These results suggest that bacterial isolates StS3 and StT2 have excellent potential to be used as effective biocontrol agents promoting plant growth with reduced disease incidence.

Effect of thiol redox state modulators on oxidative stress and sclerotial differentiation of the phytopathogenic fungus Rhizoctonia solani.

This study showed that sclerotial differentiation in the filamentous phytopathogenic fungus Rhizoctonia solani is directly related to oxidative stress and thiol redox state (TRS). Sclerotial differentiation is modulated by the availability of non-cytotoxic -SH groups as was shown by the inhibition of sclerorial differentiation by the TRS modulator N-acetyl cysteine (AcCSH), and not necessarily with those of the TRS reduced components glutathione (GSH) and its precursor cysteine (CSH) as indicated by the GSH-biosynthesis inducer and inhibitor L-2-oxo-thiazolidine-4-carboxylate and L-buthionine-S,R-sulfoximine, respectively. Moreover, inhibition of sclerotial differentiation was accompanied by decrease of the high oxidative stress indicators, lipid peroxidation and DNA damage in the mycelial substrate where sclerotia initials are formed, which suggests that this phenomenon is related to oxidative stress as it is predicted by our theory on sclerotial differentiation.

Effect of glutathione biosynthesis-related modulators on the thiol redox state enzymes and on sclerotial differentiation of filamentous phytopathogenic fungi.

In this study, sclerotial differentiation in filamentous phytopathogenic fungi, representing the four main types of sclerotia, was studied in relation to thiol redox state (TRS)-related enzymes and their substrates/products. TRS was altered by the general TRS modulator Nu-acetylcysteine (AcCSH) and by the glutathione (GSH) biosynthesis modulators L-oxo-thiazolidine-4-carboxylate (OTC), and L-buthionine-S,R-sulfoximine (BSO). This study showed that the four studied types of sclerotial differentiation are directly related with the antioxidant -SH groups of GSH and/or CSH, since the decrease of sclerotial differentiation concurred with an increase of these thiols by the GSH biosynthesis modulators AcCSH, OTC, and BSO. Supportive to that conclusion is the fact that, in general, the activities of the TRS-related enzymes GR/GPDH and Ttase decrease in the end of the undifferentiated stage due to the substitution of their antioxidant function by the antioxidant potential of the -SH group providers AcCSH and OTC. Moreover, it was found that BSO expectedly suppressed GSH biosynthesis in the tested fungi, and unexpectedly decreased their sclerotial differentiation by a dose-dependent manner typical for antioxidants. The possible antioxidant role of BSO was supported by the decrease it caused in the antioxidant enzymes GR/GPDH and Ttase. The results of this study are in accordance with our hypothesis that sclerotial differentiation in phytopathogenic fungi is induced by oxidative stress.

Biocontrol of root and crown rot in tomatoes under greenhouse conditions using Trichoderma harzianum and Paenibacillus lentimorbus: additional effect of solarization

Trichoderma harzianum 650 (Th650) and Paenebacillus lentimorbus 629 (Pl629) selected earlier for their ability to control Rhizoctonia solani, Fusarium solani and F. oxysporum in vitro, were applied alone or combined with solarization (summer assay) and/or with methyl bromide (MeBr) (summer and winter assays) to a soil with a high inoculum level, for the control of tomato root rot caused by the complex F. oxysporum f. sp. lycopersici - Pyrenochaeta lycopersici - Rhizoctonia solani. Evaluations were also performed independently for root damage caused by P. lycopersici, and also for R. solani in the summer assay. MeBr decreased tomato root damage caused by the complex from 88.7 percent to 21.2 percent and from 78.4 percent to 35.7 percent in the summer and in the winter assay, respectively. None of the bio-controllers could replace MeBr in the winter assay, but Th650 and Pl629 reduced root damage caused by this complex in the summer assay. Treatments with bio-controllers were improved by their combination with solarization in this season. Independent evaluations showed that the positive control of Th650 towards R. solani and the lack of effect on P. lycopersici correlates well with the endochitinase pattern expressed by Th650 in response to these phytopathogens. Root damage caused by R. solani can be controlled at a similar level as it does MeBr in summer assays, thus representing an alternative to the use of this chemical fungicide for the control of this phytopathogen.

Antagonism against Rhizoctonia solani and fungitoxic metabolite production by some Penicillium isolates.

A number of Penicillium isolates were recovered in association to Rhizoctonia solani strains pathogenic on tobacco and from soil on plates pre-colonized by the pathogen itself. Their antagonism toward R. solani AG-2-1 was evaluated in dual cultures in vitro. Inhibition of growth was evident to some extent in most pairings, while hyphal interactions referable to mycoparasitic relationships were not observed. However, the occurrence of plasmolysis and/or vacuolisation and the induction of monilioid cells were indicative of the release of bioactive compounds. Therefore, production of fungitoxic metabolites was tested by adding concentrated culture filtrates of each Penicillium isolate to the growth medium of R. solani. Complete and lasting inhibition was incited by culture filtrates of some isolates belonging to P. brevicompactum, P. expansum, and P. pinophilum. Three purified compounds, respectively mycophenolic acid, patulin and 3-O-methylfunicone, which were extracted from culture filtrates, were able to inhibit R. solani in vitro. Their production was also detected in dual cultures of the same Penicillium strains with R. solani prepared in sterilized soil and when the Penicillium strains were cultured directly on R. solani mycelium harvested from liquid cultures. The possible role of such metabolites in antagonism of the above-mentioned Penicillium species against R. solani is discussed.

Quinic acid induces hypovirulence and expression of a hypovirulence-associated double-stranded RNA in Rhizoctonia solani.

A double-stranded (ds)RNA, designated as M2, is associated with hypovirulence, conversion of the quinic acid pathway from inducible to constitutive and downregulation of the shikimic acid pathway in the Rhizoctonia solani culture Rhs 1A1. In this study, we report that in the virulent, M2-lacking isolate Rhs 1AP, which is isogenic to Rhs 1A1, quinic acid reduces virulence dramatically and induces synthesis of an M2-encoded polypeptide and its respective mRNA. The full-length sense strand of M2 is detected in untreated Rhs 1AP only after a second 30-cycle amplification, using nested primers. Quinate-induced Rhs 1AP contains low concentrations of both full-length sense and complementary strand of M2. The quinic acid-induced hypovirulence in Rhs 1AP cannot be overturned by the end-product of the shikimic acid pathway, chorismic acid, which enhances the virulence of Rhs 1AP dramatically when used alone. In addition to its apparent applications, this study confirms the strong association between the M2 dsRNA and hypovirulence in R. solani.

Expression of a hypovirulence-causing double-stranded RNA is associated with up-regulation of quinic acid pathway and down-regulation of shikimic acid pathway in Rhizoctonia solani.

We reported previously that a 3.6-kb double-stranded RNA, designated as M2, is associated with hypovirulence in the Rhizoctonia solani isolate Rhs 1A1 and proposed that the M2-encoded putative polypeptide A (pA) might interfere with the regulation of the quinate and shikimate pathways. In this study, Western blot analysis showed that a protein band of the predicted size (83 kDa) binds antibodies specific to a pA epitope and is detectable in M2-containing but not in M2-lacking cultures. A mRNA, associated with Rhs 1A1 polysomes immunoprecipitated with anti-pA antibodies, has a sequence basically identical to that of the sense-strand of M2. The normally inducible quinate pathway was constitutively expressed, whereas the shikimate pathway was down-regulated in the M2-containing, hypovirulent Rhs 1A1. Finally, the relative concentration of phenylalanine, precursor of the virulence determinant phenylacetic acid, was correlated with the degree of pathogenicity in the virulent Rhs 1AP but not in the hypovirulent Rhs 1A1.

Purification of angularin, a novel antifungal peptide from adzuki beans.

An antifungal peptide was isolated from the adzuki bean with a procedure involving affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. The protein designated angularin was adsorbed on both types of chromatographic media and possessed a molecular weight of 8 kDa. Angularin exhibited antifungal activity against a variety of fungal species including Mycospharella arachidiocola and Botrytis cinerea. It inhibited mycelial growth in B. cinerea with an IC50 of 14.3 microM. Fusarium oxysporum and Rhizoctonia solani were not inhibited. Angularin demonstrated inhibitory activity on translation in the rabbit reticulocyte lysate system (IC50 = 8.0 microM) but did not affect proliferation of splenocytes. The activity of HIV-1 reverse transcriptase was inhibited in the presence of angularin. Its N-terminal sequence was GEPGQKE.

BE-23372M, a novel protein tyrosine kinase inhibitor. I. Producing organism, fermentation, isolation and biological activities.

BE-23372M, a novel protein tyrosine kinase inhibitor, was isolated from the culture broth of a fungus. The producing strain, F23372, was identified as Rhizoctonia solani, based on the cultural and morphological characteristics. The active principle was extracted from the mycelium with acetone and purified by solvent extraction, silica gel column chromatography and Sephadex LH-20 column chromatography. BE-23372M showed strong inhibitory activity against EGF receptor kinase with IC50 values of 0.02 and 0.03 microM on two different substrates, whereas IC50 values against protein kinase C and cAMP-dependent protein kinase were 4.5 and > 20 microM, respectively. The compound inhibited the growth of A431 human epidermoid carcinoma and MKN-7 human stomach cancer cell lines with IC50 values of 8 and 24 microM, respectively.

Regulation of L-phenylalanine ammonia-lyase from Rhizoctonia solani.

Maximal levels of L-henylalanine ammonia-lyase activity were observed when the mycelial felts of Rhizoctonia solani were grown for 4.5 days on Byrde synthetic medium containing 3.5% glucose and 0.3% L-phenylalanine, Differential centrifugation studies have indicated that the enzyme is localized in the soluble fraction. The time course of induction of L-phenylalanine ammonia-lyase activity by L-phenylalanine showed a lag period of 1 to 1.5 h and reached a maximum around 4 to 6 h after the addition of the inducer to the medium. L-Phenylalanine, L-tyrosine, and L-tryptophan were nearly equally efficient inducers of the enzyme. D-Phenylalanine was as efficient as the L-isomer, whereas D-tyrosine was a poor inducer. Light, gibberellic acid, indole 3-acetic acid, and kinetin had no effect on the induction of L-phenylalanine ammonia-lyase activity. Cycloheximide did not inhibit the uptake of amino acids by the mycelia but completely blocked the incorporation of radioactive amino acids into soluble proteins and the development of L-phenylalanine ammonia-lyase activity. Actinomycin D inhibited both the incorporation of 32P into ribonucleic acid and the enzyme activity. Conclusive evidence for de novo synthesis of L-phenylalanine ammonia-lyase was obtained by the incorporation of radioactive amino acids into the enzyme. Electrophoretic analysis of the purified preparation showed a single protein band that coincided with radioactivity and L-phenylalanine ammonia-lyase activity. Glucose and intermediates of the tricarboxylic acid cycle, like citric acid, alpha-ketoglutaric acid, and succinic acid, and the metabolites of L-phenylalanine, like o-coumaric acid, o-hydroxyphenylacetic acid, and protocatechuic acid, significantly repressed L-phenylalanine ammonia-lyase activity. The observed repression was not relieved by cyclic adenosine 5'-triphosphate.