The inducible expression of polyphenol oxidase (PPO), a presumed antiherbivore enzyme, was examined in hybrid poplar (Populus trichocarpa ϫ Populus deltoides). Following mechanical wounding simulating insect damage, PPO activity increased dramatically in wounded and unwounded leaves on wounded plants beginning at 24 and 48 h, respectively. A hybrid poplar PPO cDNA was isolated and its nucleotide sequence determined. On northern blots, PPO transcripts were detected within 8 h of wounding, and reached peak levels at 16 and 24 h in wounded and unwounded leaves, respectively. Methyl jasmonate spray and feeding by forest tent caterpillar also induced PPO expression. The induction of PPO was strongest in the youngest four leaves, which were generally avoided by caterpillars in free feeding experiments. This wound-and herbivore-induced expression of PPO in hybrid poplar supports the defensive role of this protein against insect pests.

This study aims to evaluate the antiviral role of nucleic acid-based agonists for the activation of toll-like receptor (TLR) signaling pathways, and its protective role in respiratory influenza A virus infections. TLR-3 is expressed on myeloid dendritic cells, respiratory epithelium, and macrophages, and appears to play a central role in mediating both the antiviral and inflammatory responses of the innate immunity in combating viral infections. Influenza viruses can effectively inhibit the host's ability to produce interferons, and thereby suppress the immune system's antiviral defence mechanisms. Poly ICLC is a synthetic double stranded RNA comprising of polyriboinosinic-poly ribocytidylic acid (Poly IC) stabilized with l-lysine (L) and carboxymethylcellulose (C). Poly ICLC and liposome-encapsulated Poly ICLC (LE Poly ICLC) are TLR-3 agonists and are potent inducer of interferons and natural killer cells. Intranasal pre-treatment of mice with Poly ICLC and LE Poly ICLC provided high level of protection against lethal challenge with a highly lethal avian H5N1 influenza (HPAI) strain (A/H5N1/chicken/Henan clade 2), and against lethal seasonal influenza A/PR/8/34 [H1N1] and A/Aichi/2 [H3N2] virus strains. The duration of protective antiviral immunity to multiple lethal doses of influenza virus A/PR/8/34 virus had been previously found to persist for up to 3 weeks in mice for LE Poly ICLC and 2 weeks for Poly ICLC. Similarly, pre-treatment of mice with CpG oligonucleotides (TLR-9 agonist) was also found to provide complete protection against influenza A/PR/8/34 infection in mice. RT-PCR analysis of lung tissues of mice treated with Poly ICLC and LE Poly ICLC revealed upregulation of TLR-3 mRNAs gene expression. Taken together, these results do support the potential role of TLR-3 and TLR-9 agonists such as Poly ICLC and LE Poly ICLC in protection against lethal seasonal and HPAI virus infection.

Western equine encephalitis virus (WEEV) is a positive-sense, single-stranded RNA virus which is transmitted to equines and humans through mosquito bites. WEEV infects the central nervous system with severe complications and even death. There are no human vaccine and antiviral drugs. We investigated whether adenovirus-mediated expression of interferon alpha could be used for pre- and post-exposure protection against a lethal WEEV challenge in mice. A human adenoviral vector (Ad5-mIFNalpha) expressing mouse interferon alpha was constructed. We found that Ad5-mIFNalpha provided 100% protection against various WEEV strains in mice after a single intramuscular inoculation at 24 h, 48 h or 1 week before the challenge. When given as a single inoculation at 6 h after the challenge, Ad5-mIFNalpha delayed the progress of WEEV infection and provided about 60% protection. Our findings suggest that adenovirus-mediated expression of interferon alpha can be an alternative approach for the prevention and treatment of WEEV infection.

As part of an ongoing effort to identify genes involved in poplar defense responses, and to provide a resource for comparative analysis of woody and non-woody plant defense, we generated expressed sequence tags (ESTs) from a library constructed from systemically wounded leaves of hybrid poplar (Populus trichocarpa x P. deltoides). Partial sequences were obtained from the 5' ends of 928 individual cDNAs, which could be grouped into 565 non-overlapping sequences. Of these, 447 sequences were singletons, while the remainder fell into 118 clusters containing up to 17 partially overlapping ESTs. Approximately 81% of the EST sequences showed similarity to previously described sequences in public databases. Of these, the distribution of gene functions within the EST set indicated that approximately 11% of the ESTs encode proteins potentially involved in defense or secondary metabolism, while photosynthesis and primary metabolism accounted for 45% of the expressed genes. Two types of defense proteins, Kunitz trypsin inhibitors and chitinases, were found among the ten most abundant ESTs, indicating the significant impact of wounding on the leaf transcriptome and suggesting that these functions are important for hybrid poplar defense. In the course of this work, three new wound-inducible Kunitz trypsin inhibitor-like genes and two new chitinase-like genes were characterized. A suite of other systemically wound-induced genes were identified using northern and macroarray analysis, indicating diversity and multiplicity in the induced defense response. Overall, we demonstrate that defense-related genes of hybrid poplar have a variety of functions, and show remarkably diverse expression patterns upon wounding.