Heterologous signals allow efficient targeting of a nuclear-encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots (Arabidopsis, broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.

Archaea in the foregut of macropod marsupials: PCR and amplicon sequence-based observations

Aims To investigate, using culture-independent techniques, the presence and diversity of methanogenic archaea in the foregut of kangaroos. Methods and Results DNA was extracted from forestomach contents of 42 kangaroos (three species), three sheep and three cattle. Four qualitative and quantitative PCR assays targeting the archaeal domain (16S rRNA gene) or the functional methanogenesis gene, mcrA, were used to determine the presence and population density of archaea in kangaroos and whether they were likely to be methanogens. All ruminal samples were positive for archaea, produced PCR product of expected size, contained high numbers of archaea and high numbers of cells with mcrA genes. Kangaroos were much more diverse and contradictory. Fourteen kangaroos had detectable archaea with numbers 10- to 1000-fold fewer than sheep and cattle. Many kangaroos that did not possess archaea were positive for the mcrA gene and had detectable numbers of cells with this gene and vice versa. DNA sequence analysis of kangaroos' archaeal 16S rRNA gene clones show that many methanogens were related to Methanosphaera stadmanae. Other sequences were related to non-methanogenic archaea (Thermoplasma sp.), and a number of kangaroos had mcrA gene sequences related to methane oxidising archaea (ANME). Conclusions Discrepancies between qualitative and quantitative PCR assays for archaea and the mcrA gene suggest that the archaeal communities are very diverse and it is possible that novel species exist. Significance and Impact of the Study Archaea (in general) were below detectable limits in many kangaroos, especially Red kangaroos; when present they are in lower numbers than in ruminants, and the archaea are not necessarily methanogenic. The determination of why this is the case in the kangaroo foregut could assist in reducing emissions from other ecosystems in the future.

Discovery of genes associated with fruit ripening in arica papaya using expressed sequence tags

To identify genes involved in papaya fruit ripening, a total of 1171 expressed sequence tags (ESTs) were generated from randomly selected clones of two independent fruit cDNA libraries derived from yellow and red-fleshed fruit varieties. The most abundant sequences encoded: chitinase, 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, catalase and methionine synthase, respectively. DNA sequence comparisons identified ESTs with significant similarity to genes associated with fruit softening, aroma and colour biosynthesis. Putative cell wall hydrolases, cell membrane hydrolases, and ethylene synthesis and regulation sequences were identified with predicted roles in fruit softening. Expressed papaya genes associated with fruit aroma included isoprenoid biosynthesis and shikimic acid pathway genes and proteins associated with acyl lipid catabolism. Putative fruit colour genes were identified due to their similarity with carotenoid and chlorophyll biosynthesis genes from other plant species.

Host range, symptom expression and RNA 3 sequence analyses of six Australian strains of Cucumber mosaic virus

We have characterised six Australian Cucumber mosaic virus (CMV) strains belonging to different subgroups, determined by the sequence of their complete RNA 3 and by their host range and the symptoms they cause on species in the Solanaceae, Cucurbitaceae and on sweet corn. These data allowed classification of strains into the known three CMV subgroups and identification of plant species able to differentiate the Australian strains by symptoms and host range. Western Australian strains 237 and Twa and Queensland strains 207 and 242 are closely related members of CMV subgroup IA, which cause similar severe symptoms on Nicotiana species. Strains 207 and 237 (subgroup IA) were the only strains tested which systemically infected sweet corn. Strain 243 caused the most severe symptoms of all strains on Nicotiana species, tomato and capsicum and appears to be the first confirmed subgroup IB strain reported in Australia. Based on pair-wise distance analysis and phylogeny of RNA 3, as well as mild disease symptoms on Nicotiana species, CMV 241 was assigned to subgroup II, as the previously described Q-CMV and LY-CMV.

Discovery of genes associated with fruit ripening in Carica papaya using expressed sequence tags

To identify genes involved in papaya fruit ripening, a total of 1171 expressed sequence tags (ESTs) were generated from randomly selected clones of two independent fruit cDNA libraries derived from yellow and red-fleshed fruit varieties. The most abundant sequences encoded:chitinase, 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, catalase and methionine synthase, respectively. DNA sequence comparisons identified ESTs with significant similarity to genes associated with fruit softening, aroma and colour biosynthesis. Putative cell wall hydrolases, cell membrane hydrolases, and ethylene synthesis and regulation sequences were identified with predicted roles in fruit softening. Expressed papaya genes associated with fruit aroma included isoprenoid biosynthesis and shikimic acid pathway genes and proteins associated with acyl lipid catabolism. Putative fruit colour genes were identified due to their similarity with carotenoid and chlorophyll biosynthesis genes from other plant species.

Phylogeography of the pharaoh cuttle Sepia pharaonis based on partial mitochondrial 16S sequence data

The pharaoh cuttle Sepia pharaonis Ehrenberg, 1831 (Mollusca: Cephalopoda: Sepiida) is a broadly distributed species of substantial fisheries importance found from east Africa to southern Japan. Little is known about S. pharaonis phylogeography, but evidence from morphology and reproductive biology suggests that Sepia pharaonis is actually a complex of at least three species. To evaluate this possibility, we collected tissue samples from Sepia pharaonis from throughout its range. Phylogenetic analyses of partial mitochondrial 16S sequences from these samples reveal five distinct clades: a Gulf of Aden/Red Sea clade, a northern Australia clade, a Persian Gulf/Arabian Sea clade, a western Pacific clade (Gulf of Thailand and Taiwan) and an India/Andaman Sea clade. Phylogenetic analyses including several Sepia species show that S. pharaonis sensu lato may not be monophyletic. We suggest that "S. pharaonis" may consist of up to five species, but additional data will be required to fully clarify relationships within the S. pharaonis complex.

Host-delivered RNAi: An effective strategy to silence genes in plant parasitic nematodes

Root-knot nematodes (Meloidogyne spp.) are obligate, sedentary endoparasites that infect many plant species causing large economic losses worldwide. Available nematicides are being banned due to their toxicity or ozone-depleting properties and alternative control strategies are urgently required. We have produced transgenic tobacco (Nicotiana tabacum) plants expressing different dsRNA hairpin structures targeting a root-knot nematode (Meloidogyne javanica) putative transcription factor, MjTis11. We provide evidence that MjTis11 was consistently silenced in nematodes feeding on the roots of transgenic plants. The observed silencing was specific for MjTis11, with other sequence-unrelated genes being unaffected in the nematodes. Those transgenic plants able to induce silencing of MjTis11, also showed the presence of small interfering RNAs. Even though down-regulation of MjTis11 did not result in a lethal phenotype, this study demonstrates the feasibility of silencing root-knot nematode genes by expressing dsRNA in the host plant. Host-delivered RNA interference-triggered (HD-RNAi) silencing of parasite genes provides a novel disease resistance strategy with wide biotechnological applications. The potential of HD-RNAi is not restricted to parasitic nematodes but could be adapted to control other plant-feeding pests.

Three new Lasiodiplodia spp. from the tropics, recognized based on DNA sequence comparisons and morphology

Botryosphaeria rhodina (anamorph Lasiodiplodia theobromae) is a common endophyte and opportunistic pathogen on more than 500 tree species in the tropics and subtropics. During routine disease surveys of plantations in Australia and Venezuela several isolates differing from L. theobromae were identified and subsequently characterized based upon morphology and ITS and EF1-a nucleotide sequences. These isolates grouped into three strongly supported clades related to but different from the known taxa, B. rhodina and L. gonubiensis, These have been described here as three new species L. venezuelensis sp. nov., L. crassispora sp. nov. and L. rubropurpurea sp. nov. The three could be distinguished easily from each other and the two described species of Lasiodiplodia, thus confirming phylogenetic separations. Furthermore all five Lasiodiplodia spp. now recognized separated from Diplodia spp. and Dothiorella spp. with 100% bootstrap support.

Detection of Chlamydiaceae DNA in veterinary specimens using a family-specific PCR

Aims: The aim of this work was to develop a rapid molecular test for the detection of the Chlamydiaceae family, irrespective of the species or animal host. Methods and Results: The method described herein is a polymerase chain reaction targeting the 16S rRNA gene of the Chlamydiaceae family, and the results demonstrate that the test reacts with five reference Chlamydiaceae but none of the 19 other bacterial species or five uninfected animal tissues tested. The results also indicate the enhanced sensitivity of this test when compared with conventional culture or serology techniques. This is demonstrated through parallel testing of six real clinical veterinary cases and confirmatory DNA sequence analysis. Conclusions, Significance and Impact of the Study: This test can be used by veterinary diagnostic laboratories for rapid detection of Chlamydiaceae in veterinary specimens, with no restriction of chlamydial species or animal host. The test does not differentiate chlamydial species, and if required, speciation must be carried out retrospectively using alternate methods. However, for the purpose of prescribing therapy for chlamydiosis, this test would be an invaluable laboratory tool.

Australian and Thai isolates of Burkholderia pseudomallei are distinct by multilocus sequence typing: Revision of a case of mistaken identity

A recent study using multilocus sequence typing (MLST) of Burkholderia pseudomallei isolates found a sequence type (ST60) to be common to both Thailand and Australia, contradicting earlier studies showing complete distinction between isolates from these regions. The ST60 isolates reportedly from Australia had been obtained for MLST from United Kingdom and U.S. collections. We have located and characterized the original Australian isolates; they were collected in 1983, and they are neither ST60 nor B. pseudomallei isolates. The B. pseudomallei MLST database has been corrected, and there is no ST common to isolates verified as obtained from Australia or from Thailand.

Completion of the genome sequence of Lettuce necrotic yellows virus, type species of the genus Cytorhabdovirus

We completed the genome sequence of Lettuce necrotic yellows virus (LNYV) by determining the nucleotide sequences of the 4a (putative phosphoprotein), 4b, M (matrix protein), G (glycoprotein) and L (polymerase) genes. The genome consists of 12,807 nucleotides and encodes six genes in the order 3′ leader-N-4a(P)-4b-M-G-L-5′ trailer. Sequences were derived from clones of a cDNA library from LNYV genomic RNA and from fragments amplified using reverse transcription-polymerase chain reaction. The 4a protein has a low isoelectric point characteristic for rhabdovirus phosphoproteins. The 4b protein has significant sequence similarities with the movement proteins of capillo- and trichoviruses and may be involved in cell-to-cell movement. The putative G protein sequence contains a predicted 25 amino acids signal peptide and endopeptidase cleavage site, three predicted glycosylation sites and a putative transmembrane domain. The deduced L protein sequence shows similarities with the L proteins of other plant rhabdoviruses and contains polymerase module motifs characteristic for RNA-dependent RNA polymerases of negative-strand RNA viruses. Phylogenetic analysis of this motif among rhabdoviruses placed LNYV in a group with other sequenced cytorhabdoviruses, most closely related to Strawberry crinkle virus.

Phylogenetic relationships of graminicolous downy mildews based on cox2 sequence data

Graminicolous downy mildews (GDM) are an understudied, yet economically important, group of plant pathogens, which are one of the major constraints to poaceous crops in the tropics and subtropics. Here we present a first molecular phylogeny based on cox2 sequences comprising all genera of the GDM currently accepted, with both lasting (Graminivora, Poakatesthia, and Viennotia) and evanescent (Peronosclerospora, Sclerophthora, and Sclerospora) sporangiophores. In addition, all other downy mildew genera currently accepted, as well as a representative sample of other oomycete taxa, have been included. It was shown that all genera of the GDM have had a long, independent evolutionary history, and that the delineation between Peronosclerospora and Sclerospora is correct. Sclerophthora was found to be a particularly divergent taxon nested within a paraphyletic Phytophthora, but without support. The results confirm that the placement of Peronosclerospora and Sclerospora in the Saprolegniomycetidae is incorrect. Sclerophthora is not closely related to Pachymetra of the family Verrucalvaceae, and also does not belong to the Saprolegniomycetidae, but shows close affinities to the Peronosporaceae. In addition, all GDM are interspersed throughout the Peronosporaceae s lat., suggesting that a separate family for the Sclerosporaceae might not be justified.

Ruminococcus bromii, identification and isolation as a dominant community member in the rumen of cattle fed a barley diet

Aims: To identify dominant bacteria in grain (barley)-fed cattle for isolation and future use to increase the efficiency of starch utilization in these cattle. Methods and Results: Total DNA was extracted from samples of the rumen contents from eight steers fed a barley diet for 9 and 14 days. Bacterial profiles were obtained using denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V2/V3 region of the 16S rRNA genes from total bacterial DNA. Apparently dominant bands were excised and cloned, and the clone insert sequence was determined. One of the most common and dominant bacteria present was identified as Ruminococcus bromii. This species was subsequently isolated using traditional culture-based techniques and its dominance in the grain-fed cattle was confirmed using a real-time Taq nuclease assay (TNA) designed for this purpose. In some animals, the population of R. bromii reached densities above 1010R. bromii cell equivalents per ml or approximately 10% of the total bacterial population. Conclusions: Ruminococcus bromii is a dominant bacterial population in the rumen of cattle fed a barley-based diet. Significance and Impact of the Study: Ruminococcus bromii YE282 may be useful as a probiotic inoculant to increase the efficiency of starch utilization in barley-fed cattle. The combination of DGGE and real-time TNA has been an effective process for identifying and targeting for isolation, dominant bacteria in a complex ecosystem.

Simple sequence repeat markers associated with three quantitative trait loci for black point resistance can be used to enrich selection populations in bread wheat

Black point in wheat has the potential to cost the Australian industry $A30.4 million a year. It is difficult and expensive to screen for resistance, so the aim of this study was to validate 3 previously identified quantitative trait loci (QTLs) for black point resistance on chromosomes 2B, 4A, and 3D of the wheat variety Sunco. Black point resistance data and simple sequence repeat (SSR) markers, linked to the resistance QTLs and suited to high-throughput assay, were analysed in the doubled haploid population, Batavia (susceptible) × Pelsart (resistant). Sunco and Pelsart both have Cook in their pedigree and both have the Triticum timopheevii translocation on 2B. SSR markers identified for the 3 genetic regions were gwm319 (2B, T. timopheevii translocation), wmc048 (4AS), and gwm341 (3DS). Gwm319 and wmc048 were associated with black point resistance in the validation population. Gwm341 may have an epistatic influence on the trait because when resistance alleles were present at both gwm319 and wmc048, the Batavia-derived allele at gwm341 was associated with a higher proportion of resistant lines. Data are presented showing the level of enrichment achieved for black point resistance, using 1, 2, or 3 of these molecular markers, and the number of associated discarded resistant lines. The level of population enrichment was found to be 1.83-fold with 6 of 17 resistant lines discarded when gwm319 and wmc048 were both used for selection. Interactions among the 3 QTLs appear complex and other genetic and epigenetic factors influence susceptibility to black point. Polymorphism was assessed for these markers within potential breeding material. This indicated that alternative markers to wmc048 may be required for some parental combinations. Based on these results, marker-assisted selection for the major black point resistance QTLs can increase the rate of genetic gain by improving the selection efficiency and may facilitate stacking of black point resistances from different sources.

Diversity of methanogens in ruminants in Queensland

Methane emissions from ruminant livestock represent a loss of carbon during feed conversion, which has implications for both animal productivity and the environment because this gas is considered to be one of the more potent forms of greenhouses gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, a thorough knowledge of the diversity of these microbes in different breeds of cattle and sheep, as well as in response to different diets, is required. A study was undertaken using the molecular techniques denaturing gradient gel electrophoresis, DNA cloning and DNA sequence analysis to define the extent of diversity among methanogens in ruminants, particularly Bos indicus cross cattle, on differing forages in Queensland. It was found that the diversity of methanogens in forage-fed cattle in Queensland was greater than in grain-fed cattle but there was little variability in methanogen community composition between cattle fed different forages. The species that dominate the rumen microbial communities of B. indicus cross cattle are from the genus Methanobrevibacter, although rumen-fluid inoculated digestors fed Leucaena leucocephala leaf were populated with Methanosphaera-like strains, with the Methanobrevibacter-like strains displaced. If ruminant methane emissions are to be reduced, then antimethanogen bioactives that target both broad groups of ruminant methanogens are most likely to be needed, and as a part of an integrated suite of approaches that redirect rumen fermentation towards other more useful end products.