The coleopteran insect western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte) is a devastating crop pest in North America and Europe. Although crop plants that produce Bacillus thuringiensis (Bt) proteins can limit insect infestation, some insect populations have evolved resistance to Bt proteins. Here we describe an insecticidal protein, designated IPD072Aa, that is isolated from Pseudomonas chlororaphis. Transgenic corn plants expressing IPD072Aa show protection from WCR insect injury under field conditions. IPD072Aa leaves several lepidopteran and hemipteran insect species unaffected but is effective in killing WCR larvae that are resistant to Bt proteins produced by currently available transgenic corn. IPD072Aa can be used to protect corn crops against WCRs.

It is well established that the expression of Bacillus thuringiensis (B.t.) toxin genes in higher plants is severely limited at the mRNA level, but the cause remains controversial. Elucidating whether mRNA accumulation is limited transcriptionally or posttranscriptionally could contribute to effective gene design as well as provide insights about endogenous plant gene-expression mechanisms. To resolve this controversy, we compared the expression of an A/Urich wild-type cryIA(c) gene and a G/C-rich synthetic cryIA(c) B.t.-toxin gene under the control of identical 5 and 3 flanking sequences. Transcriptional activities of the genes were equal as determined by nuclear run-on transcription assays. In contrast, mRNA half-life measurements demonstrated directly that the wildtype transcript was markedly less stable than that encoded by the synthetic gene. Sequences that limit mRNA accumulation were located at more than one site within the coding region, and some appeared to be recognized in Arabidopsis but not in tobacco (Nicotiana tabacum). These results support previous observations that some A/U-rich sequences can contribute to mRNA instability in plants. Our studies further indicate that some of these sequences may be differentially recognized in tobacco cells and Arabidopsis.Gene transfer into plants has become a relatively simple and routine process in many species. However, successful integration of a transgene into the plant genome does not automatically result in expression. For example, introduction of an additional copy of an endogenous gene can trigger cosuppression mechanisms, resulting in silencing of both the introduced and the endogenous genes (for review, see Meyer and Saedler, 1996). In the case of foreign genes transferred into plants, it is not uncommon to find that the introduced gene is incompatible with plant geneexpression mechanisms. Bacillus thuringiensis (B.t.)-toxin genes are perhaps the most widely known example of foreign genes for which it has been difficult to obtain useful levels of expression in transgenic plants (Diehn et al., 1996). Although a variety of mechanisms controlling gene expression have been blamed for poor B.t.-toxin expression, the exact cause remains controversial because of the limited amount of data gathered to address the problem. This is in part due to the inherent difficulties associated with studying a gene expressed at very low levels. It is also due to the fact that, for practical applications in agriculture, this problem can be circumvented by the brute-force approach of constructing highly modified synthetic versions of B.t.-toxin genes that yield high levels of expression. Nevertheless, elucidating the mechanisms that limit B.t.-toxin expression could make the design of new genes more efficient and provide insight about steps in gene expression that are relevant to endogenous plant genes.A widely observed characteristic of B.t.-toxin gene expression in plants has been that little or no mRNA accumulates, even when transcription is under the control of strong pr...

-toxin-coding region corresponding to the short transcripts. A fourth polyadenylation site was identified using a chimeric gene. These results demonstrate for the first time to our knowledge that premature polyadenylation can limit the expression of a foreign gene in plants. Moreover, this work emphasizes that further study of the fundamental principles governing polyadenylation in plants will have basic as well as applied significance.

Maize and Arabidopsis root apical meristems differ in several aspects of their radial organization and ontogeny. Despite the large evolutionary distance and differences in root radial patterning, analysis of the putative maize ortholog of the Arabidopsis patterning gene SCARECROW ( SCR ) revealed expression localized to the endodermis, which is similar to its expression in Arabidopsis. Expression in maize extends through the quiescent center, a population of mitotically inactive cells formerly thought to be undifferentiated and to lack radial pattern information. Zea mays SCARECROW ( ZmSCR ), the putative maize SCR ortholog, was used as a molecular marker to investigate radial patterning during regeneration of the root tip after either whole or partial excision. Analysis of the dynamic expression pattern of ZmSCR as well as other markers indicates the involvement of positional information as a primary determinant in regeneration of the root radial pattern.

Maize and Arabidopsis root apical meristems differ in several aspects of their radial organization and ontogeny. Despite the large evolutionary distance and differences in root radial patterning, analysis of the putative maize ortholog of the Arabidopsis patterning gene SCARECROW ( SCR ) revealed expression localized to the endodermis, which is similar to its expression in Arabidopsis. Expression in maize extends through the quiescent center, a population of mitotically inactive cells formerly thought to be undifferentiated and to lack radial pattern information. Zea mays SCARECROW ( ZmSCR ), the putative maize SCR ortholog, was used as a molecular marker to investigate radial patterning during regeneration of the root tip after either whole or partial excision. Analysis of the dynamic expression pattern of ZmSCR as well as other markers indicates the involvement of positional information as a primary determinant in regeneration of the root radial pattern.

Antibodies directed to polysaccharide (PS) antigens of bacteria are crucial to host immunity to infection. The isotypes of antibodies made to PS, however, are restricted primarily to IgM and IgG1 and IgG2 in man and to IgM and IgG3 in mice. Using sequential sublining and sib selection, an IgG1 murine monoclonal antibody that has variable regions identical to those of a parent IgG3 monoclonal antibody directed to the high-molecular-weight component of the O-specific side chain of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide was derived. These antibodies differed markedly in their antigen binding and effector functions. IgG3 was superior in binding to multivalent PS both in purified and whole bacterial form, fixation of the third component of complement to the bacterial surface, and opsonization of P. aeruginosa for uptake by both murine and human phagocytes. These data suggest that the IgG subclass of these murine anti-LPS antibodies is an important determinant of both avidity for multivalent antigen and biologic function.

Crops expressing Bacillus thuringiensis (Bt)-derived insecticidal protein genes have been commercially available for over 15 years and are providing significant value to growers. However, there remains the need for alternative insecticidal actives due to emerging insect resistance to certain Bt proteins. A screen of bacterial strains led to the discovery of a two-component insecticidal protein named AfIP-1A/1B from an Alcaligenes faecalis strain. This protein shows selectivity against coleopteran insects including western corn rootworm (WCR). Transgenic maize plants expressing AfIP-1A/1B demonstrate strong protection from rootworm injury. Surprisingly, although little sequence similarity exists to known insecticidal proteins, efficacy tests using WCR populations resistant to two different Cry proteins show that AfIP-1A/1B and mCry3A differ in their mode of action while AfIP-1A/1B and the binary Cry34Ab1/Cry35Ab1 protein share a similar mode. These findings are supported by results of competitive binding assays and the similarity of the x-ray structure of AfIP-1A to Cry34Ab1. Our work indicates that insecticidal proteins obtained from a non-Bt bacterial source can be useful for developing genetically modified crops and can function similarly to familiar proteins from Bt.

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