Overview of worldwide diversity of Diaphorina citri Kuwayama mitochondrial cytochrome oxidase 1 haplotypes: two Old World lineages and a New World invasion.

Relationships among worldwide collections of Diaphorina citri (Asian citrus psyllid) were analyzed using mitochondrial cytochrome oxidase I (mtCOI) haplotypes from novel primers. Sequences were produced from PCR amplicons of an 821bp portion of the mtCOI gene using D. citri specific primers, derived from an existing EST library. An alignment was constructed using 612bps of this fragment and consisted of 212 individuals from 52 collections representing 15 countries. There were a total of eight polymorphic sites that separated the sequences into eight different haplotypes (Dcit-1 through Dcit-8). Phylogenetic network analysis using the statistical parsimony software, TCS, suggests two major haplotype groups with preliminary geographic bias between southwestern Asia (SWA) and southeastern Asia (SEA). The recent (within the last 15 to 25 years) invasion into the New World originated from only the SWA group in the northern hemisphere (USA and Mexico) and from both the SEA and SWA groups in the southern hemisphere (Brazil). In only one case, Reunion Island, did haplotypes from both the SEA and SWA group appear in the same location. In Brazil, both groups were present, but in separate locations. The Dcit-1 SWA haplotype was the most frequently encountered, including ~50% of the countries sampled and 87% of the total sequences obtained from India, Pakistan and Saudi Arabia. The second most frequently encountered haplotype, Dcit-2, the basis of the SEA group, represented ~50% of the countries and contained most of the sequences from Southeast Asia and China. Interestingly, only the Caribbean collections (Puerto Rico and Guadeloupe) represented a unique haplotype not found in other countries, indicating no relationship between the USA (Florida) and Caribbean introductions. There is no evidence for cryptic speciation for D. citri based on the COI region included in this study.

First Report of "Candidatus Liberibacter psyllaurous" Associated with Psyllid Yellows of Tomato in Colorado.

Greenhouse tomato growers from Fort Lupton, CO contacted the USDA-ARS-USHRL in 2002 regarding plant symptoms resembling "psyllid yellows" associated with Bactericera cockerelli (Sulc) infestations that initially begin as retarded growth, erectness of new growth, chlorosis, and purpling of leaves followed by widespread chlorosis and production of many small, poor-quality fruit (1). Symptoms appeared ≈6 weeks after psyllids were observed and were generally restricted to the top half of the plant. Leaf cuttings from beefsteak tomatoes cv. Quest were immediately placed in RNAlater (Applied Biosystems, Austin, TX). Samples from symptomatic and asymptomatic plants were collected in September and December of 2002. At each date, leaves were sampled from multiple plants and placed in separate RNAlater bottles. September samples exhibited initial "psyllid yellows" symptoms and December samples exhibited severe symptoms. Samples remained at 4°C in RNAlater for 6 years until recent findings suggested that a new species of bacteria, named either "Candidatus Liberibacter psyllaurous" (2) or "Ca. L. solanacearum" (3), may be the causal agent of "psyllid yellows". The Qiagen (Valencia, CA) DNeasy Plant Kit and recommended protocols were used for four separate DNA isolations from each of the four tomato samples that had previously remained unopened. Five PCR primer pairs designed to amplify three distinct genetic regions within the "Ca L. psyllaurous" rrn operon (16S rRNA, 16S-23S rRNA intergenic region, and 23S rRNA) were used and one primer pair specific to the tomato DNA (18S rRNA gene) that successfully amplified from all samples was used as a positive control. Bacterial primers included one pair designed specifically for 16S rRNA sequences of 'Ca. L. asiaticus', 'americanus', and 'africanus' species (USHRL-CL1) and four sets, Lp-1 through Lp-4, previously described (2) that amplify nonoverlapping regions of the 16S-23S rRNA operon. The USHRL-CL1 primers (USHRL-CL1f: 5'-CTTACCAGCCCTTGACATGTATAGGA-3', and USHRL-CL1r: 5'-TCCCTATAAAGTACCCAACATCTAGGTAAA-3') amplify a 195-bp fragment from bp 895 to 1,089 of the 'Ca. Liberibacter' sp. 16S rRNA Genbank Accession No. L22532. Only samples from severe symptomatic plants collected in December 2002 yielded amplicons that were purified and sequenced (Genbank: USHRL-CL1, FJ871062; Lp-1, FJ871058; Lp-2, FJ871059; Lp-3, FJ871060; Lp-4, FJ871061). For each bacterial primer pair, the fragment amplified was highly homologous (98 to 100% identity) to "Ca. L. psyllaurous" rRNA gene/intergenic space sequences. The 16S rRNA coding region was identical to two GenBank 'Ca. Liberibacter' sp. entries: EU921627 and EU921626 from B. cockerelli samples collected in Dalhart, TX and zebra chip potato samples from Garden City, KS, respectively; however, the whole 2,500 bp amplified and sequenced from our sample contained 11 to 14 polymorphisms when compared to nine "Ca. L. psyllaurous" sequences. Our results clearly indicate that "Ca. L. psyllaurous" isolates were associated with tomato "psyllid yellows" symptoms in Colorado as early as 2002 and significant sequence variation exists within the 16S/23S rRNA intergenic region and 23S rRNA coding region to allow analysis of genetic diversity among "Ca. L. psyllaurous" isolates. References: (1) L. B. Daniels. Ph.D. diss. University of Minnesota, St. Paul, 1954. (2) A. K. Hansen et al. Appl. Environ. Microbiol. 74:5862, 2008. (3) L. W. Liefting et al. Plant Dis. 93:208, 2009.

Analysis of host preference and geographical distribution of Anastrepha suspensa (Diptera: Tephritidae) using phylogenetic analyses of mitochondrial cytochrome oxidase I DNA sequence data.

Anastrepha suspensa (Loew) is an economically important pest, restricted to the Greater Antilles and southern Florida. It infests a wide variety of hosts and is of quarantine importance in citrus, a multi-million dollar industry in Florida. The observed recent increase in citrus infested with A. suspensa in Florida has raised questions regarding host-specificity of certain populations and genetic diversity of the pest throughout its geographical distribution. Cytochrome oxidase I (COI) DNA sequence data was used to characterize the genetic diversity of A. suspensa from Florida and Caribbean populations reared from different host plants. Maximum likelihood and Bayesian phylogenetic methods were used to analyse COI data. Sequence variation among mitochondrial COI genes from 107 A. suspensa samples collected throughout Florida and the Caribbean ranged between 0 and 10% and placed all A. suspensa as a monophyletic group that united all A. suspensa in a clade sister to a Central American group of the A. fraterculus paraphyletic species complex. The most likely tree of the COI locus indicated that COI sequence variation was too low to provide resolution at the subspecies level, therefore monophyletic groups based on host-plant use, geography (Florida, Jamaica, Cayman Islands, Puerto Rico or Dominican Republic) or population sampled are not supported. This result indicates that either no population segregation has occurred based on these biological or geographical distinctions and that this is a generalist, polyphagous invasive genotype. Alternatively, if populations are distinct, the segregation event was more recent than can be distinguished based on COI sequence variation.

Aphid biology: expressed genes from alate Toxoptera citricida, the brown citrus aphid.

The brown citrus aphid, Toxoptera citricida (Kirkaldy), is considered the primary vector of citrus tristeza virus, a severe pathogen which causes losses to citrus industries worldwide. The alate (winged) form of this aphid can readily fly long distances with the wind, thus spreading citrus tristeza virus in citrus growing regions. To better understand the biology of the brown citrus aphid and the emergence of genes expressed during wing development, we undertook a large-scale 5' end sequencing project of cDNA clones from alate aphids. Similar large-scale expressed sequence tag (EST) sequencing projects from other insects have provided a vehicle for answering biological questions relating to development and physiology. Although there is a growing database in GenBank of ESTs from insects, most are from Drosophila melanogaster and Anopheles gambiae, with relatively few specifically derived from aphids. However, important morphogenetic processes are exclusively associated with piercing-sucking insect development and sap feeding insect metabolism. In this paper, we describe the first public data set of ESTs from the brown citrus aphid, T. citricida. The cDNA library was derived from alate adults due to their significance in spreading viruses (e.g., citrus tristeza virus). Over 5180 cDNA clones were sequenced, resulting in 4263 high-quality ESTs. Contig alignment of these ESTs resulted in 2124 total assembled sequences, including both contiguous sequences and singlets. Approximately 33% of the ESTs currently have no significant match in either the non-redundant protein or nucleic acid databases. Sequences returning matches with an E-value of < or = -10 using BLASTX, BLASTN, or TBLASTX were annotated based on their putative molecular function and biological process using the Gene Ontology classification system. These data will aid research efforts in the identification of important genes within insects, specifically aphids and other sap feeding insects within the Order Hemiptera.

Characterizing the interaction between fire ants (Hymenoptera: Formicidae) and developing soybean plants.

This research characterizes the interaction between the fire ants Solenopsis invicta Buren and developing soybean plants. Phagostimulant studies showed that fire ant foraging on soybean seeds increased once the seeds imbibed water. During seedling development over a 5-d germination period, fire ant foraging shifted from the stem/cotyledons to the roots, despite continual increases in fresh weights for each region, and the fact that stem/cotyledon tissue contained the majority of food reserves. Carbohydrate analysis showed that although 2-d-old seedlings had higher concentrations of phagostimulant carbohydrates, especially sucrose, than tissues of mature plants, all tissues analyzed had enough of these sugars to induce a phagostimulant response. Fire ant association with seeds/seedlings germinated in soil resulted in reduced seedling vigor, as determined by a doubling of seedling emergence time, a threefold increase in malformed seedlings, and visible damage to cotyledons. Seeds germinated and grown to mature plants in association with fire ants, allocated 43% more assimilate into pods, but produced 28% less root dry matter, 11% less total dry matter, and there was an 81% reduction in the number of root nodules compared with control plants. We propose that reduced root development and inhibitions of nodule formation would be major yield limiting factors under field conditions. This work demonstrates that fire ant damage to soybeans is not limited to seedling establishment and that more research should be directed at the subterranean activities of the fire ant.

Regulation of the maize ubiquitin (Ubi-1) promoter in developing maize (Zea mays L.) seeds examined using transient gene expression in kernels grown in vitro.

Kernel culture was assessed for evaluating novel gene expression in developing maize (Zea mays L.) seeds by comparing the transient expression of maize ubiquitin (Ubi-1) promoter-driven ß-glucuronidase (GUS) delivered by particle bombardment in kernels grown in culture with those grown in planta. With kernels from either source, GUS expression, as determined by histochemical staining, was widespread in young, actively growing kernels, but it diminished with kernel age and by 25 days after pollination was found only in the embryo. Transient expression of Ubi-1 in kernels grown in vitro was not affected by wounding, ethylene treatment, pathogen invasion, or heat shock. In contrast, the plant hormones indole-3-acetic acid and kinetin both stimulated transient Ubi-1 expression in the endosperm, particularly at the periphery. Transient gene expression in developing maize seeds grown in vitro should allow for facile and rapid evaluation of the tissue-specificity and environmental responses of novel gene constructs in developing maize seeds.

Isolation and characterization of a novel glutamine synthetase from Rhizobium meliloti.

Two glutamine synthetases, GSI and GSII, are found in most rhizobia. However, WSU650, a Rhizobium meliloti glnA glnII mutant that lacks both enzymes, can grow without a glutamine supplement in minimal medium that contains both ammonium and glutamate. The bacteria contained a third glutamine synthetase, GSIII, which has been purified and partially characterized. GSIII had considerable glutamine synthetase activity when assayed using a semibiosynthetic (glutamate- and hydroxylamine-dependent) assay, but had no detectable transferase (glutamine- and hydroxyl-amine-dependent) activity. GSIII was inhibited by ADP and pyrophosphate but not by various nitrogen-containing metabolites that inhibit other GS enzymes. Activity was also inhibited by methionine sulfoximine, a transition state analog, but the concentration needed to inhibit GSIII was 50 to 100 times higher than that needed to inhibit GSI or GSII. GSIII had a Km for glutamate of 13.3 mM, for ammonium of 33 mM, and for hydroxylamine of 5.3 mM with a pH optimum of 6.8 and a temperature optimum of 50 degrees C. The purified protein had related subunits of 46.5 and 49 kDa and a native molecular mass of 355 kDa, indicating the native enzyme was an octamer. Polyclonal antibodies specific for GSIII reacted with a protein of similar molecular weight in Escherichia coli strains that carry R. meliloti glnT on a plasmid. GSIII activity was detected in some of these strains that contained glnT. Extracts of root nodules formed by WSU650 also react with the antibodies.

The 70-Kilodalton Heat Shock Cognate Can Act as a Molecular Chaperone during the Membrane Translocation of a Plant Secretory Protein Precursor.

When a model secretory precursor was synthesized in vitro and analyzed by rate-zonal sedimentation, it appeared to be associated with other proteins present in a wheat germ extract. At least one of the associated proteins is a member of the 70-kD family of stress proteins. It was possible to immunoprecipitate the secretory precursor with anti-heat shock cognate 70 (Hsc70) antibodies in the absence but not in the presence of ATP, suggesting that the association was specific. ATP-sensitive association is one diagnostic characteristic of molecular chaperone-type proteins. Increasing incubation temperature decreased the amount of precursor associated with Hsc70. A method was developed for the removal of Hsc70 from a wheat germ in vitro translation mixture by immunoprecipitation. Cotranslational translocation and processing of the secretory precursor by maize endosperm microsomes were inefficient in the Hsc70-depleted system but were greatly stimulated by addition of purified preparations of various heat shock 70 proteins (Hsp70s). Cytosolic Hsc70 from maize endosperm was capable of autophosphorylation in vitro. Phosphorylated Hsc70 was much less efficient in promoting membrane translocation of the secretory precursor. These results suggest that chaperone function in vivo could be regulated by phosphorylation.

A zein signal sequence functions as a signal-anchor when fused to maize alcohol dehydrogenase.

A chimeric gene, preZad, was constructed encoding a zein signal sequence fused precisely to the amino terminus of maize alcohol dehydrogenase 1. Translocation and processing of this chimeric preZad protein were assayed in vitro using a rabbit reticulocyte lysate translation system supplemented with canine pancreatic microsomes. PreZad was cotranslationally translocated across the vesicular membranes. Unexpectedly, the signal sequence was not removed although a suitable cleavage site was preserved and presented within the vesicle lumen. Failure to cleave the signal sequence was apparently not due to the lack of a beta-turn near the processing site. When a beta-turn was introduced near the cleavage site through site-directed mutagenesis, no processing was observed. PreZad was not solubilized by alkaline treatment of the microsomes, indicating an integral membrane association. Resistance to proteolysis, in the absence of detergent, indicates that preZad is associated with the membranes in a type II orientation (C-terminus in and N-terminus outside the vesicles). Analysis of truncated versions of preZad showed that it is the uncleaved signal sequence that functions as a signal-anchor. Changing the ratio of net charge flanking the signal sequence to less than 1 (N-terminal:C-terminal) did not alter the type II membrane orientation, as would have been predicted by the 'positive-in rule'. Our results provide additional insight into the role of the passenger protein and signal sequence-flanking regions in recognition of a signal peptidase processing site, and the orientation of insertion of a signal-anchor sequence into the endoplasmic reticulum membrane.

Glutamine synthetase II in Rhizobium: reexamination of the proposed horizontal transfer of DNA from eukaryotes to prokaryotes.

We have determined the DNA sequence of a Rhizobium meliloti gene that encodes glutamine synthetase II (GSII). The deduced amino acid sequence was compared to that of Bradyrhizobium japonicum GSII and those of various plant and mammalian glutamine synthetases (GS) in order to evaluate a proposal that the gene for this enzyme was recently transferred from plants to their symbiotic bacteria. There is 83.6% identity between the R. meliloti and B. japonicum proteins. The bacterial GSII proteins average 42.5% identity with the plant GS proteins and 41.8% identity with their mammalian counterparts. The plant proteins average 53.7% identity with the mammalian proteins. Thus, the GS proteins are highly conserved and the divergence of these proteins is proportional to the phylogenetic divergence of the organisms from which the sequences were determined. No transfer of genes across large taxonomic gaps is needed to explain the presence of GSII in these bacteria.

Isolation, characterization, and complementation of Rhizobium meliloti 104A14 mutants that lack glutamine synthetase II activity.

The glutamine synthetase (GS)-glutamate synthase pathway is the primary route used by members of the family Rhizobiaceae to assimilate ammonia. Two forms of glutamine synthetase, GSI and GSII, are found in Rhizobium and Bradyrhizobium species. These are encoded by the glnA and glnII genes, respectively. Starting with a Rhizobium meliloti glnA mutant as the parent strain, we isolated mutants unable to grow on minimal medium with ammonia as the sole nitrogen source. For two auxotrophs that lacked any detectable GS activity, R. meliloti DNA of the mutated region was cloned and partially characterized. Lack of cross-hybridization indicated that the cloned regions were not closely linked to each other or to glnA; they therefore contain two independent genes needed for GSII synthesis or activity. One of the cloned regions was identified as glnII. An R. meliloti glnII mutant and an R. meliloti glnA glnII double mutant were constructed. Both formed effective nodules on alfalfa. This is unlike the B. japonicum-soybean symbiosis, in which at least one of these GS enzymes must be present for nitrogen-fixing nodules to develop. However, the R. meliloti double mutant was not a strict glutamine auxotroph, since it could grow on media that contained glutamate and ammonia, an observation that suggests that a third GS may be active in this species.

Regulation of glutamine synthetase II activity in Rhizobium meliloti 104A14.

Most rhizobia contain two glutamine synthetase (GS) enzymes: GSI, encoded by glnA, and GSII, encoded by glnII. We have found that WSU414, a Rhizobium meliloti 104A14 glutamine auxotroph derived from a glnA parental strain, is an ntrA mutant. The R. meliloti glnII promoter region contains DNA sequences similar to those found in front of other genes that require ntrA for their transcription. No GSII was found in the glnA ntrA mutant, and when a translational fusion of glnII to the Escherichia coli lacZ gene was introduced into WSU414, no beta-galactosidase was expressed. These results indicate that ntrA is required for glnII expression. The ntrA mutation did not prevent the expression of GSI. In free-living culture, the level of GSII and of the glnII-lacZ fusion protein was regulated by altering transcription in response to available nitrogen. No GSII protein was detected in alfalfa, pea, or soybean nodules when anti-GSII-specific antiserum was used.