Functional Annotation of Hypothetical Proteins Derived from Suppressive Subtraction Hybridization (SSH) Analysis Shows NPR1 (Non-Pathogenesis Related)-Like Activity

Fusarium wilt is considered the most devastating banana disease incited by Fusarium oxysporum f. sp. cubense (FOC). The present study addresses suppressive subtraction hybridization (SSH) analysis for differential gene expression in banana plant, mediated through FOC and its interaction with biocontrol agent Trichoderma asperellum (prr2). SSH analysis yielded a total of 300 clones. The resultant clones were sequenced and processed to obtain 22 contigs and 87 singleton sequences. BLAST2GO (Basic Local Alignment Search Tool 2 Gene Ontology) analysis was performed to assign known protein function. Initial functional annotation showed that contig 21 possesses p38-like endoribonuclease activity and duality in subcellular localization. To gain insights into its additional roles and precise functions, a sequential docking protocol was done to affirm its role in the defense pathway. Atomic contact energies revealed binding affinities in the order of miRNA > phytoalexins > polyubiquitin, emphasizing their role in the Musa defense pathway. Contig 21 and polyubiquitin showed an atomic contact energy value of −479.60 kJ/mol, and even higher atomic contact energies were observed for miRNA (−804.86, −482.28, −494.75 kJ/mol), demonstrating its high RNA-binding properties. Phytoalexin contig 21-interacting interfacial residues were identified as rigid (10)/non-rigid (2) based on Bi, N values, and B-factor per residue. Hence, based on these results, contig 21 was characterized as a NPR1 (non-pathogenesis-related protein) homolog that is involved in plant defense and systemic induced resistance.

Inhibition of radiation-induced glioblastoma invasion by genetic and pharmacological targeting of MDA-9/Syntenin

Glioblastoma multiforme (GBM) is an intractable tumor despite therapeutic advances, principally because of its invasive properties. Radiation is a staple in therapeutic regimens, although cells surviving radiation can become more aggressive and invasive. Subtraction hybridization identified melanoma differentiation-associated gene 9 [MDA-9/Syntenin; syndecan-binding protein (SDCBP)] as a differentially regulated gene associated with aggressive cancer phenotypes in melanoma. MDA-9/Syntenin, a highly conserved double-PDZ domain-containing scaffolding protein, is robustly expressed in human-derived GBM cell lines and patient samples, with expression increasing with tumor grade and correlating with shorter survival times and poorer response to radiotherapy. Knockdown of MDA-9/Syntenin sensitizes GBM cells to radiation, reducing postradiation invasion gains. Radiation induces Src and EGFRvIII signaling, which is abrogated through MDA-9/Syntenin down-regulation. A specific inhibitor of MDA-9/Syntenin activity, PDZ1i (113B7), identified through NMR-guided fragment-based drug design, inhibited MDA-9/Syntenin binding to EGFRvIII, which increased following radiation. Both genetic (shmda-9) and pharmacological (PDZ1i) targeting of MDA-9/Syntenin reduced invasion gains in GBM cells following radiation. Although not affecting normal astrocyte survival when combined with radiation, PDZ1i radiosensitized GBM cells. PDZ1i inhibited crucial GBM signaling involving FAK and mutant EGFR, EGFRvIII, and abrogated gains in secreted proteases, MMP-2 and MMP-9, following radiation. In an in vivo glioma model, PDZ1i resulted in smaller, less invasive tumors and enhanced survival. When combined with radiation, survival gains exceeded radiotherapy alone. MDA-9/Syntenin (SDCBP) provides a direct target for therapy of aggressive cancers such as GBM, and defined small-molecule inhibitors such as PDZ1i hold promise to advance targeted brain cancer therapy.

Profiling of differentially expressed genes in ectomycorrhizal fungus Pisolithus tinctorius responding to mycorrhiza helper Brevibacillus reuszeri MPt17

A cDNA library of the ectomycorrhizal (ECM) fungus Pisolithus tinctorius Pt2 after interaction with the mycorrhiza helper bacterium (MHB) Brevibacillus reuszeri MPt17 was constructed by suppression subtraction hybridization. Total RNA from B. reuszeri MPt17 exudates treated Pi. tinctorius Pt2 was used as a “tester” and total RNA from nonbacterial treated Pi. tinctorius Pt2 was used as a “driver.” Among the differentially expressed sequences, a BLASTX in the NCBI non-redundant protein sequence database revealed that 75% of the non-redundant sequences (147 out of 196) were highly similar to known proteins (E-value < e−5). Twelve sequences were annotated as mycelium development function combining with a potential functional categories using gene ontology. Quantitative real-time PCR analysis showed that all of the 3 symbiosis regulated acidic polypeptide genes were all up-regulated in the MPt17-treated Pt2. These results provide evidence that the MHB B. reuszeri MPt17 could significantly change the expression of symbiosis-related genes and genes in mycelium development in ECM fungus, and also support the hypothesis that the MHB functions as helper though promotion on fungal mycelium.