Multitrophic interactions of the silverleaf whitefly,host plants,competing herbivores,and phytopathogens

Our laboratory found that silverleaf whitefly (SLW; Bemisia argentifolii Bellows & Perring) feeding alters host plant physiology and chemistry. The SLW induces a number of host plant defenses, including pathogenesis-related (PR) protein accumulation (e.g., chitinases, beta-1,3-glucanases, peroxidases, chitosanases, etc.). Induction of the PR proteins by SLW feeding occurs in various plant species and varieties. The extent and type of induction is dependent on a number of factors that include host plant growing conditions, the length of time the host plant is exposed to SLW feeding, the plant variety, and SLW population densities. The appearance of PR proteins correlates well with reduced infestations of conspecific insect herbivore competitors. Greenhouse and field experiments in which herbivore competitors (cabbage looper, Trichoplusia ni; leaf miner, Liromyza trifolii) were placed on plants previously exposed to SLW feeding demonstrated behavioral differences (oviposition, feeding preferences) and reduced survival rates and development times of these insects. The interaction was asymmetrical, i.e., SLW infestations of plants previously exposed to leaf miners had little or no effect on SLW behavior (oviposition). Induction of plant-defensive proteins by SLW feeding was both local (at the feeding site) and systemic (uninfested leaves distant to the feeding site). There are interactions between diseases such as tomato mottle virus (ToMoV; a geminivirus) and the host plant and SLW. PR proteins were induced in tomato plants infected with ToMoV much as they were via non-viruliferous SLW feeding. The presence of ToMoV in tomato plants significantly increased the number of eggs produced by SLW females. Experiments using tomato plants, powdery mildew (PM), and tobacco mosaic virus (TMV) show that whitefly infestations can affect plant pathogen relationships but the effects vary among pathogen types. Enzyme analyses prior to pathogen inoculation showed that whitefly treatment significantly increased the activities of foliar chitinase and peroxidase. Evaluation of pathogen growth 3 weeks after inoculation showed that whitefly feeding significantly reduced the incidence of PM. However, TMV levels evaluated by ELISA were not significantly affected by whitefly feeding. Six weeks after inoculation with pathogens, the chitinase and peroxidase activities were still elevated in plants initially fed on by whiteflies but continuing pathogen infection had no effect on these enzymes. The possibility that geminivirus infection and/or SLW infestations isolate the host plant for the selected reproduction of the virus and the insect is discussed. Multitrophic cascade effects may contribute to the successful eruptive appearance of SLW on various crops, ranking them as a major pest. They may explain the general observation that when SLW infest a host plant there are few if any competing insect herbivores and pathogens found in the host. However, the results indicate that certain SLW-virus relationships could be mutualistic.     

Intact microtubules support adenovirus and herpes simplex virus infections

Capsids and the enclosed DNA of adenoviruses, including the species C viruses adenovirus type 2 (Ad2) and Ad5, and herpesviruses, such as herpes simplex virus type 1 (HSV-1), are targeted to the nuclei of epithelial, endothelial, fibroblastic, and neuronal cells. Cytoplasmic transport of fluorophore-tagged Ad2 and immunologically detected HSV-1 capsids required intact microtubules and the microtubule-dependent minus-end-directed motor complex dynein-dynactin. A recent study with epithelial cells suggested that Ad5 was transported to the nucleus and expressed its genes independently of a microtubule network. To clarify the mechanisms by which Ad2 and, as an independent control, HSV-1 were targeted to the nucleus, we treated epithelial cells with nocodazole (NOC) to depolymerize microtubules and measured viral gene expression at different times and multiplicities of infections. Our results indicate that in NOC-treated cells, viral transgene expression was significantly reduced at up to 48 h postinfection (p.i.). A quantitative analysis of subcellular capsid localization indicated that NOC blocked the nuclear targeting of Ad2 and also HSV-1 by more than 90% at up to 7 h p.i. About 10% of the incoming Texas Red-coupled Ad2 (Ad2-TR) was enriched at the nucleus in microtubule-depleted cells at 5 h p.i. This result is consistent with earlier observations that Ad2-TR capsids move randomly in NOC-treated cells at less than 0.1 micro m/s and over distances of less than 5 micro m, characteristic of Brownian motion. We conclude that fluorophore-tagged Ad2 and HSV-1 particles are infectious and that microtubules play a prominent role in efficient nuclear targeting during entry and gene expression of species C Ads and HSV-1.     

Structural investigation of the binding of a herpesviral protein to the SH3 domain of tyrosine kinase Lck

Herpesvirus saimiri codes for a tyrosine kinase interacting protein (Tip) that interacts with both the SH3 domain and the kinase domain of the T-cell-specific tyrosine kinase Lck via two separate motifs. The activation of Lck by Tip is considered as a key event in the transformation of human T-lymphocytes during herpesviral infection. We investigated the interaction of proline-rich Tip peptides with the LckSH3 domain starting with the structural characterization of the unbound interaction partners. The solution structure of the LckSH3 was determined by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy using 44 residual dipolar couplings in addition to the conventional experimental restraints. Circular dichroism spectroscopy proved that the polyproline helix of Tip is already formed prior to SH3 binding and is conformationally stable. NMR titration experiments point out three major regions of the Tip-Lck interaction comprising the RT loop, the n-src loop, and a helical turn preceding the last strand of the beta-sheet. Further changes of the chemical shifts were observed for the N- and C-terminal beta-strands of the SH3 domain, indicating additional contacts outside the proline-rich segment or subtle structural rearrangements transmitted from the binding site of the proline helix. Fluorescence spectroscopy shows that Tip binds to the SH3 domains of several Src kinases (Lck, Hck, Lyn, Src, Fyn, Yes), exhibiting the highest affinities for Lyn, Hck, and Lck.     

Bansformation of tobacco with a mutated cyanobacterial phytoene desaturase gene confers resistance to bleaching herbicides

Carotenoids are constituents of the photosynthetic apparatus and essential for plant survival because of their involvement in protection of chlorophylls against photooxidation. Certain classes of herbicides are interfering with carotenoid biosynthesis leading to pigment destruction and a bleached plant phenotype. One important target site for bleaching herbicides is the enzyme phytoene desaturase catalysing the desaturation of phytoene in zeta-carotene. This enzymatic reaction can be inhibited by norflurazon or fluridone. We have transformed tobacco with a mutated cyanobacterial phytoene desaturase gene (pds) derived from the Synechococcus PCC 7942 mutant NFZ4. Characterization of the resulting transformants revealed an up to 58 fold higher norflurazon resistance in comparison to wild type controls. The tolerance for fluridone was also increased 3 fold in the transgenics. Furthermore, the transformed tobacco maintained a higher level of D1 protein of photosystem II indicating a lower susceptibility to photooxidative damage in the presence of norflurazon. In contrast, the genetic manipulation did not confer herbicide resistance against zeta-carotene desaturase inhibitors.     

Vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans: FTIR and electrochemical studies on Tyr-D4-labeled and on Tyr280His and Tyr35Phe mutant enzymes

A combined electrochemical and FTIR spectroscopic approach was used to identify the vibrational modes of tyrosines in cytochrome c oxidase from Paracoccus denitrificans which change upon electron transfer and coupled proton transfer. Electrochemically induced FTIR difference spectra of the Tyr-D4-labeled cytochrome c oxidase reveal that only small contributions arise from the tyrosines. Contributions between 1600 and 1560 cm(-1) are attributed to nu8a/8b(CC) ring modes. The nu19(CC) ring mode for the protonated form of tyrosines is proposed to absorb with an uncommonly small signal at 1525-1518 cm(-1) and for the deprotonated form at 1496-1486 cm(-1), accompanied by the increase of the nu19(CC) ring mode of the Tyr-D(4)-labeled oxidase at approximately 1434 cm(-1). A signal at 1270 cm(-1) can be tentatively attributed to the nu7'a(CO) and delta(COH) mode of a protonated tyrosine. Uncommon absorptions, like the mode at 1524 cm(-1), indicate the involvement of Tyr280 in the spectra. Tyr280 is a crucial residue close to the binuclear center and is covalently bonded to His276. The possible changes of the spectral properties are discussed together with the absorbance spectra of tyrosine bound to histidine. The vibrational modes of Tyr280 are further analyzed in combination with the mutation to histidine, which is assumed to abolish the covalent bonding. The electrochemically induced FTIR difference spectra of the Tyr280His mutant point to a change in protonation state in the environment of the binuclear center. Together with an observed decrease of a signal at 1736 cm(-1), previously assigned to Glu278, a possible functional coupling is reflected. In direct comparison to the FTIR difference spectra of the D4-labeled compound and comparing the spectra at pH 7 and 4.8, the protonation state of Tyr280 is discussed. Furthermore, a detailed analysis of the mutant is presented, the FTIR spectra of the CO adduct revealing a partial loss of Cu(B). Electrochemical redox titrations reflect a downshift of the heme a3 midpoint potential by 95 +/- 10 mV. Another tyrosine identified to show redox dependent changes upon electron transfer is Tyr35, a residue in the proposed D-pathway of the cytochrome c oxidase.