RNA interference (RNAi) can be used to study gene function by effecting degradation of the targeted transcript. However, the effectiveness of transgene-induced RNAi among multiple target genes has not been compared systematically. To this end, we developed a relative quantitative RT-PCR protocol that allows use of a single internal standard over a wide range of target gene expression levels. Using this method in an analysis of transgenic Arabidopsis thaliana RNAi lines targeting 25 different endogenes revealed that independent, homozygous, single-copy (sc) T4 lines targeting the same gene generally reduce transcript levels to the same extent, whereas multi-copy RNAi lines differed in the degree of target reduction and never exceeded the effect of sc transgenes. The maximal reduction of target transcript levels varied among targets. These observations suggest that each target sequence possesses an inherent degree of susceptibility to dsRNA-mediated degradation.

The molecular chaperone HEAT SHOCK PROTEIN90 (HSP90) is essential for the maturation of key regulatory proteins in eukaryotes and for the response to temperature stress. Earlier, we have reported that fungi living in association with plants of the Sonoran desert produce small molecule inhibitors of mammalian HSP90. Here, we address whether elaboration of the HSP90 inhibitor monocillin I (MON) by the rhizosphere fungus Paraphaeosphaeria quadriseptata affects plant HSP90 and plant environmental responsiveness. We demonstrate that MON binds Arabidopsis (Arabidopsis thaliana) HSP90 and can inhibit the function of HSP90 in lysates of wheat (Triticum aestivum) germ. MON treatment of Arabidopsis seedlings induced HSP101 and HSP70, conserved components of the stress response. Application of MON, or growth in the presence of MON, allowed Arabidopsis wild type but not AtHSP101 knockout mutant seedlings to survive otherwise lethal temperature stress. Finally, cocultivation of P. quadriseptata with Arabidopsis enhanced plant heat stress tolerance. These data demonstrate that HSP90-inhibitory compounds produced by fungi can influence plant growth and responses to the environment.

Two new potential ligands of the Drosophila PS2 integrins have been characterized by functional interaction in cell culture. These potential ligands are a new Drosophila laminin ␣2 chain encoded by the wing blister locus and Ten-m, an extracellular protein known to be involved in embryonic pattern formation. As with previously identified PS2 ligands, both contain RGD sequences, and RGD-containing fragments of these two proteins (DLAM-RGD and TENM-RGD) can support PS2 integrin-mediated cell spreading. In all cases, this spreading is inhibited specifically by short RGD-containing peptides. As previously found for the PS2 ligand tiggrin (and the tiggrin fragment TIG-RGD), TENM-RGD induces maximal spreading of cells expressing integrin containing the ␣ PS2C splice variant. This is in contrast to DLAM-RGD, which is the first Drosophila polypeptide shown to interact preferentially with cells expressing the ␣ PS2 m8 splice variant. The ␤ PS integrin subunit also varies in the presumed ligand binding region as a result of alternative splicing. For TIG-RGD and TENM-RGD, the ␤ splice variant has little effect, but for DLAM-RGD, maximal cell spreading is supported only by the ␤ PS4A form of the protein. Thus, the diversity in PS2 integrins due to splicing variations, in combination with diversity of matrix ligands, can greatly enhance the functional complexity of PS2-ligand interactions in the developing animal. The data also suggest that the splice variants may alter regions of the subunits that are directly involved in ligand interactions, and this is discussed with respect to models of integrin structure.The integrins are a family of heterodimeric transmembrane glycoproteins, consisting of ␣ and ␤ subunits, that serve as receptors for extracellular matrix molecules and cell surface molecules of neighboring cells. Integrins have roles in diverse phenomena, such as cell adhesion, spreading, migration, and differentiation, as well as roles in the development and progression of numerous pathological states, such as cancer and cardiovascular disease (1-4). As might be expected from these varied requirements, integrins not only provide mechanical linkages to the matrix and neighboring cells but also receive and transmit information from the cell exterior to the cell interior, and vice versa (5). The fruit fly, Drosophila melanogaster, provides a valuable genetic system in which to examine these integrin functions in the developing animal (6, 7). As a complement to these genetic studies, we have utilized cultured cells, expressing various combinations of Drosophila PS integrin transgenes, to examine interactions of PS integrins and potential integrin ligands.The PS1, PS2, and PS3 integrins of Drosophila consist of a common ␤ PS subunit paired with an ␣ PS1 , ␣ PS2 , or ␣ PS3 subunit, respectively. ␤ PS , ␣ PS1 , and ␣ PS2 were originally identified as position-specific (PS) 1 antigens in monoclonal antibody screens of imaginal discs (8, 9). Subsequent biochemical and molecular analyses of these antigens indicated that they are...

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Tiggrin is a novel extracellular matrix ligand for the Drosophila PS2 integrins. We have used flanking P elements to generate a precise deletion of tiggrin. Most flies lacking tiggrin die as larvae or pupae. A few adults do emerge and these appear to be relatively normal, displaying only misshapen abdomens and a low frequency of wing defects. Examination of larvae shows that muscle connections, function and morphology are defective in tiggrin mutants. Muscle contraction waves that extend the length of the larvae are much slower in tiggrin mutants. Direct examination of bodywall muscles shows defects in muscle attachment sites, where tiggrin is specifically localized, and muscles appear thinner. Transgenes expressing tiggrin are capable of rescuing tiggrin mutant phenotypes. Transgenes expressing a mutant tiggrin, whose Arg-Gly-Asp (RGD) integrin recognition sequence has been mutated to Leu-Gly-Ala (LGA) show much reduced, but significant, rescuing ability. Cell spreading assays detect no interactions of this mutant tiggrin with PS2 integrins. Therefore, while the RGD sequence is critical for PS2 interactions and full activity in the whole fly, the mutant tiggrin retains some function(s) that are probably mediated by interactions with other ECM molecules or cell surface receptors