Fungal necrosis and ethylene inducing proteins (NEPs) induce cell death and elicit strong immune responses in many crops. In this study, we report the cloning and characterization of a transcript encoding NEP from Ganoderma boninense which belongs to a family of fungi in Ganodermataceae that cause serious infections of cacao, rubber, tea, coffee and palms. The open reading frame (ORF) encoding NEP inG. boninense(GbNEP) was cloned by 5′and 3'rapid amplification of cDNA ends (RACE) PCR. The transcript abundance of GbNEP increased in fungal culture treated with salicylic acid and jasmonic acid, respectively. The soluble recombinant Gb EP expressed in Escherichia coli BL21(DE3) pLysS was able to induce necrosis in tobacco and tomato but ineffective when applied to oil palm leaves and root tissues. The recombinant GbNEP could induce localized cell death, and production of hydrogen peroxide and superoxide in tobacco leaves. The addition of LaCl3(a calcium channel inhibitor) and EGTA (a Ca2+chelator) to tobacco leaves prior to the challenge with GbNEP significantly reduced the necrosis symptoms indicating that Ca2+is required for the action of GbNEP. In summary, this is the first report of NEP from Ganoderma species which may contribute to further research on the role of GbNEP during disease development.

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Aims: Expression of recombinant proteins across a range of different host organisms has profound contribution to the advancement in biotechnology. In this study, we aimed to construct a highly versatile broad host range (BHR) expression vector, designated as pYL101C. Methodology and results: The Golden Gate cloning approach was used to construct pYL101C. Key features of pYL101C include a strong integron promoter (PINTc), a BHR pBBR1 origin of replication (ori), gentamycin resistance gene (Gm R ) as a selectable marker and a multiple cloning site (MCS) downstream of the promoter for easy-cloning purpose. To verify the functionality of pYL101C, we cloned the superfolder green fluorescent protein (sfGFP) reporter gene into pYL101C and transferred the resultant recombinant plasmid pYL101C::sfGFP into various Gram-negative bacteria. Transformants obtained stably expressed strong green fluorescence under blue light excitation even without selection after four passages. Conclusion, significance and impact of study: The constructed BHR expression vector, pYL101C and recombinant pYL101C::sfGFP are stable and can be used to monitor the presence of Gram-negative bacteria, such as endophytes and pathogens in their hosts and environment.