Signal-induced activation of caspases, the critical protease effectors of apoptosis, requires proteolytic processing of their inactive proenzymes. Consequently, regulation of procaspase processing is critical to apoptotic execution. We report here that baculovirus pancaspase inhibitor P35 and inhibitor of apoptosis Op-IAP prevent caspase activation in vivo, but at different steps. By monitoring proteolytic processing of endogenous Sfcaspase-1, an insect group II effector caspase, we show that Op-IAP blocked the first activation cleavage at TETD2G between the large and small caspase subunits. In contrast, P35 failed to affect this cleavage, but functioned downstream to block maturation cleavages (DXXD2(G/A)) of the large subunit. Substitution of P35's reactive site residues with TETDG failed to increase its effectiveness for blocking TETD2G processing of proSf-caspase-1, despite wild-type function for suppressing apoptosis. These data are consistent with the involvement of a novel initiator caspase that is resistant to P35, but directly or indirectly inhibitable by Op-IAP. The conservation of TETD2G processing sites among insect effector caspases, including Drosophila drICE and DCP-1, suggests that in vivo activation of these group II caspases involves a P35-insensitive caspase and supports a model wherein apical and effector caspases function through a proteolytic cascade to execute apoptosis in insects.The caspases are critical protease mediators of apoptosis and thus represent important targets for anti-apoptotic intervention (reviewed in Refs. 1-4). These highly conserved, aspartate-specific proteases are expressed as single-chain zymogens, which upon apoptotic signaling are activated by proteolytic processing, either by autoactivation, transactivation, or cleavage by other caspases (reviewed in Refs. 5 and 6). Once activated, the caspases proteolytically cleave a multitude of cellular substrates, leading to apoptotic death. Thus, caspase activation is a key regulatory point in the commitment to apoptosis.The molecular mechanisms regulating caspase activation are largely unknown. In mammals, a proteolytic cascade is initiated by group III caspases with long N-terminal prodomains (reviewed in Refs. 2, 3, 6, and 7). Upon activation, initiator caspases proteolytically cleave group II effector caspases at aspartate-containing sites through a regulated sequence of reactions that separate the large and small subunit, and then detach the short prodomain (reviewed in Ref. 4). To date, it is unknown whether analogous cascades are conserved in other organisms, including insects. Nevertheless, diverse apoptotic inhibitors from mammals, insects, and their associated viral pathogens are providing important insight into the regulatory mechanisms of caspase activation (8 -14).The baculoviruses encode two mechanistically distinct apoptotic suppressors, inhibitor of apoptosis (IAP) 1 and P35. Both viral proteins prevent premature insect cell death and thereby promote virus multiplication (reviewed in Ref. 15). The baculovirus...

Xylella fastidiosa, the causal agent of several scorch diseases, is associated with leaf scorch symptoms in Chitalpa tashkentensis, a common ornamental landscape plant used throughout the southwestern United States. For a number of years, many chitalpa trees in southern New Mexico and Arizona exhibited leaf scorch symptoms, and the results from a regional survey show that chitalpa trees from New Mexico, Arizona, and California are frequently infected with X. fastidiosa. Phylogenetic analysis of multiple loci was used to compare the X. fastidiosa infecting chitalpa strains from New Mexico, Arizona, and trees imported into New Mexico nurseries with previously reported X. fastidiosa strains. Loci analyzed included the 16S ribosome, 16S-23S ribosomal intergenic spacer region, gyrase-B, simple sequence repeat sequences, X. fastidiosa-specific sequences, and the virulence-associated protein (VapD). This analysis indicates that the X. fastidiosa isolates associated with infected chitalpa trees in the Southwest are a highly related group that is distinct from the four previously defined taxons X. fastidiosa subsp. fastidiosa (piercei), X. fastidiosa subsp. multiplex, X. fastidiosa subsp. sandyi, and X. fastidiosa subsp. pauca. Therefore, the classification proposed for this new subspecies is X. fastidiosa subsp. tashke.

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Bean golden mosaic virus (BGMV) is a whitefly-transmitted, ssDNA geminivirus with a bipartite genome. AC1 is the only ORF required for geminiviral replication. A putative NTP-binding motif, EGX4GKTX32DD, was present in the derived amino acid sequence of the replication-associated protein from the AC1 ORF for 13 geminiviruses including BGMV-GA (Guatemalan isolate, amino acids 221-263). We analyzed the phenotypes of mutations within this domain using a rapid and sensitive PCR-based assay for geminiviral replication developed for these studies. Replication in tobacco cells (NT-1 suspension cells) and infection of beans were abolished when codons were changed from K228 to H or D262 to R within the putative NTP-binding site. A temperature-sensitive replication phenotype was conferred by changing E221 to R within the putative NTP-binding domain. Replication was unaffected by changing a nonconserved codon near the putative NTP-binding domain from 1190 to R. Our results demonstrate that the putative NTP-binding domain is required for geminiviral replication. The role of NTP hydrolysis and the possible value of these mutants in a trans-dominant interference scheme for virus-derived resistance are discussed.

As red imported fire ant, Solenopsis invicta Buren, continues to expand its range into the southwestern United States, it can be easily confused with the native southern fire ant, Solenopsis xyloni McCook. Variability in the morphological characteristics commonly used to differentiate these ant species was quantified by examining the length of the clypeal tooth, striations of the mesopleuron, length of antennal scape, area of the petiolar process, number and size of mandibular teeth, and color by using both scanning electron and light microscopy. Given enough samples, the average values of each of these characteristics is different between the two species; however, significant morphological variability occurs in both S. xyloni and S. invicta populations, creating an area of overlap where either of the two species could exhibit similar characteristics. Better differentiation of these two species is achieved using a combination of characteristics, but morphological techniques are not dependable unless numerous ants from each population are analyzed by a taxonomist familiar with Solenopsis. For situations requiring a more accurate identification, such as before quarantining a county or a portion of a county, a molecular technique using mitochondrial DNA and polymerase chain reaction techniques was developed.

Strain distribution for the alfalfa weevil, Hypera postica (Gyllenhal), was determined for the five major alfalfa-growing regions of New Mexico. Mitochondrial DNA from field-collected weevils at each location was digested and sequenced to differentiate among strains. Eastern, Egyptian, and western strains were recovered, along with a population not matching any known strain. The majority of sites examined had populations of two strains intermingled within a field. This is the first confirmation of the presence of all three strains within the same geographic region. Possible implications for alfalfa weevil management are discussed.

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