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Two Soybean mosaic virus (SMV) G2 isolates, L and L-RB, sharing high-sequence similarly but differing in ability to break Rsv4-mediated resistance in soybean, were investigated. Infectious clones corresponding to these two isolates and their chimeric clones resulting from swapping different regions of genomic cDNA between L and L-RB were constructed. Only L-RB or chimeras containing the middle fragment of L-RB cDNA showed virulence on Rsv4-genotype soybean. Sequence comparison analysis revealed that the middle genomic region of L and L-RB encodes four different amino acids. Point mutagenesis demonstrated that a single amino acid substitution (Q1033K) in the P3 protein determined virulence toward Rsv4 resistance. In addition, six new SMV Rsv4 resistance-breaking isolates, variants of the second passage on Williams 82 infected with the chimeras or mutants noninfectious on soybean carrying Rsv4, were obtained. Sequencing data indicated that these new isolates contain either the Q1033K mutation or a new substitution (G1054R) in P3. Site-directed mutagenesis confirmed the virulence role of the G1054R mutation on Rsv4-genotype soybean. Taken together, these data suggest that P3 of the SMV G2 strain is an avirulent determinant for Rsv4 and one single nucleotide mutation in P3 may be sufficient to compromise its elicitor function.

Until recently, low accumulation levels have been the major bottleneck for plant-made recombinant protein production. However, several breakthroughs have been described in the past few years allowing for very high accumulation levels, mainly through chloroplast transformation and transient expression, coupled with subcellular targeting and protein fusions. Another important factor influencing our ability to use plants for the production of recombinant proteins is the availability of quick and simple purification strategies. Recent developments using oleosin, zein, ELP and hydrophobin fusion tags have shown promise as efficient and cost-effective methods for non-chromatographic separation. Furthermore, plant glycosylation is a major barrier to the parenteral administration of plant-made biopharmaceuticals because of potential immunogenicity concerns. A major effort has been invested in humanizing plant glycosylation, and several groups have been able to reduce or eliminate immunogenic glycans while introducing mammalian-specific glycans. Finally, biosafety issues and public perception are essential for the acceptance of plants as bioreactors for the production of proteins. Over recent years, it has become clear that food and feed plants carry an inherent risk of contaminating our food supply, and thus much effort has focused on the use of non-food plants. Presently, Nicotiana benthamiana has emerged as the preferred host for transient expression, while tobacco is most frequently used for chloroplast transformation. In this review, we focus on the main issues hindering the economical production of recombinant proteins in plants, describing the current efforts for addressing these limitations, and we include an extensive list of recent patents generated with the intention of solving these limitations.

Great advances have been made in our understanding of the host plant's common symbiosis functions, which in legumes mediate intracellular accommodation of both nitrogen-fixing bacteria and arbuscular mycorrhiza (AM) fungi. However, it has become apparent that additional plant genes are required specifically for bacterial entry inside the host root. In this opinion article, we consider Lotus japonicus nap1 and pir1 symbiotic mutants within the context of other deleterious mutations that impair an intracellular accommodation of bacteria but have no impact on the colonization of roots by AM fungi. We highlight a clear delineation of early signaling events during bacterial versus AM symbioses while suggesting a more intricate origin of the plant's ability for intracellular accommodation of bacteria., (Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.)

Waterlogging is a serious impediment to crop productivity worldwide which acts to reduce oxygen levels in the rhizosphere due to the low diffusion rate of molecular oxygen in water. Plants respond to low oxygen through rapid and specific changes at both the transcriptional and translational levels. Transcriptional changes to low-oxygen (hypoxia) stress have been studied in a number of plant species using whole genome microarrays. Using transcriptome data from root tissue from early time points (4-5 h) from cotton (Gossypium hirsutum), Arabidopsis and gray poplar (Populus x canescens), we have identified a core set of orthologous genes that responded to hypoxia in similar ways between species, and others that showed species specific responses . Responses to hypoxia were most similar between Arabidopsis and cotton, while the waterlogging tolerant poplar species exhibited some significant differences.

Intercellular transport of viruses through cytoplasmic connections, termed plasmodesmata (PD), is essential for systemic infection in plants by viruses. Previous genetic and ultrastructural data revealed that the potyvirus cyclindrical inclusion (CI) protein is directly involved in cell-to-cell movement, likely through the formation of conical structures anchored to and extended through PD. In this study, we demonstrate that plasmodesmatal localization of CI in N. benthamiana leaf cells is modulated by the recently discovered potyviral protein, P3N-PIPO, in a CI:P3N-PIPO ratio-dependent manner. We show that P3N-PIPO is a PD-located protein that physically interacts with CI in planta. The early secretory pathway, rather than the actomyosin motility system, is required for the delivery of P3N-PIPO and CI to PD. Moreover, CI mutations that disrupt virus cell-to-cell movement compromise PD-localization capacity. These data suggest that the CI and P3N-PIPO complex coordinates the formation of PD-associated structures that facilitate the intercellular movement of potyviruses in infected plants.

A deficiency in major seed storage proteins is associated with a nearly two-fold increase in sulfur amino acid content in genetically related lines of common bean (Phaseolus vulgaris). Their mature seed proteome was compared by an approach combining label-free quantification by spectral counting, 2-DE, and analysis of selective extracts. Lack of phaseolin, phytohemagglutinin and arcelin was mainly compensated by increases in legumin, alpha-amylase inhibitors and mannose lectin FRIL. Along with legumin, albumin-2, defensin and albumin-1 were major contributors to the elevated sulfur amino acid content. Coordinate induction of granule-bound starch synthase I, starch synthase II-2 and starch branching enzyme were associated with minor alteration of starch composition, whereas increased levels of UDP-glucose 4-epimerase were correlated with a 30% increase in raffinose content. Induction of cell division cycle protein 48 and ubiquitin suggested enhanced ER-associated degradation. This was not associated with a classical unfolded protein response as the levels of ER HSC70-cognate binding protein were actually reduced in the mutant. Repression of rab1 GTPase was consistent with decreased traffic through the secretory pathway. Collectively, these results have implications for the nutritional quality of common bean, and provide information on the pleiotropic phenotype associated with storage protein deficiency in a dicotyledonous seed., (Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.)

Pear decline (PD) is a serious disease of pear (Pyrus communis L.) caused by 'Candidatus Phytoplasma pyri', which belongs to the subgroup 16SrX-C of the apple proliferation (AP) group of phytoplasmas (3). Pear seedlings from the Agriculture and Agri-Food Canada (AAFC) pear breeding program, which have been selected for advanced test and grower trials, are routinely submitted to the Canadian Food Inspection Agency (CFIA) Sidney Laboratory (formerly, CFIA Centre for Plant Health, Saanichton, BC) for virus testing at the same time that propagation is initiated to produce trees for further evaluations. In early 2007, the CFIA reported that samples of two seedling selections submitted in 2005 tested positive for phytoplasmas by a nested PCR assay with phytoplasma universal primers P1/P7 (1), followed by phytoplasma universal primers fU5/rU3 (2) and real time PCR with universal phytoplasma primers developed by the CFIA-Sidney (personal communication). Phytoplasmas present in both selections were subsequently identified as 'Ca. P. pyri' strains by nested PCR with the P1/P7 primers followed by PD/peach yellow leaf roll (PYLR)-specific primers fPD/rPDS (2,4). These were the first PD-positive results from many samples submitted over the years for testing. Following PD-positive diagnoses for the seedling trees, others propagated from these seedling trees were removed from the nursery. When tested by PD-specific nested PCR (P1/P7 then fPD/rPDS), one selection had 39 of 79 nursery trees (49%) that were PD positive, while the other selection had 27 of 96 trees (28%) testing as PD positive. PCR amplification of DNA isolated from leaves of six of the propagated trees, with primer pair fPD/rPDS, yielded an ~1,400-bp product that was sequenced. A consensus sequence of 1,313 bp (GenBank Accession No. GU565959) was subjected to a nucleotide BLAST search of the NCBI database and showed 100% nt identity with sequences of phytoplasmas PD1 (AJ542543) and PYLR (Y16394). Subsequently, the PD-positive results from leaf, dormant shoot, and root tissues from the original seedling trees were confirmed by PD-specific nested PCR. On the original seedling trees, visible symptoms typical of PD, especially premature leaf coloration, were observed in late summer 2008 and samples taken of green and red leaves were subjected to PD-specific PCR. Red leaves were PD-positive, while green leaves were mostly PD-negative. Pear leaves, dormant shoots, and roots collected from research and commercial orchards in southern Ontario in 2007 and 2008 were subjected to PD-specific nested PCR (P1/P7 then fPD/rPDS), AP-specific nested PCR (P1/P7 then fO1/rO1) (2), as well as the universal phytoplasma nested PCR (P1/P7 then fU5/rU3), resulting in the identification of PD-positive trees of several cultivars. The sequence of the 1,057-bp amplicon from accession PYR0190 (selection HW615), with AP-specific primers fO1/rO1, was deposited in GenBank (GU475131). Although there have been no previous reports of PD in Ontario, Canada, it would appear that PD has been present for some time based on the number and distribution (both geographic and cultivar) of positive samples. References: (1) S. Deng and C. Hiruki. J. Microbiol. Methods 14:53, 1991. (2) K.-H. Lorenz et al. Phytopathology 85:771, 1995. (3) E. Seemüller et al. J. Plant Pathol. 80:3, 1998. (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

The up-regulation of plant defense-related toxins or metabolic enzyme binding proteins often leads to a negative effect on herbivorous insects. These negative effects can manifest themselves at three points: changes in food ingestion, post-ingestive-changes, and post-digestive changes. Many studies have related the decrease in herbivore growth and/or survival with expression of chemicals that interfere with post-digestive effects such as the anti-nutritive effects of protease inhibitors. Nevertheless, it is unclear whether such compounds impact herbivores via earlier ingestive processes. We addressed this question by using a jasmonate-deficient mutant (Def-1), a jasmonate-overexpressor mutant (Prosystemin or Prosys), and wild-type tomato in three trials with 5th instar Trichoplusia ni. Decreases in relative growth rate (RGR) confirmed that T. ni fed on the Prosys plants developed poorly compared to those feeding on Def-1 plants (larvae on wild-types were intermediate). Preingestive and postingestive processes contributed to this effect. Total food ingested and the consumptive index were 25% lower on Prosys plants compared to Def-1 plants. Post-ingestive processes, measured by approximate digestibility, were 62% greater on Prosy plants. Post-digestive efficiency measured by conversion of ingested and digested food (ECI and ECD) decreased on Prosys plants two-fold compared to Def-1 plants. This post-digestive interference correlated well with the 2-fold decrease in activity of digestive enzymes, serine proteases, in Prosys-fed T. ni compared to those on Def-1 plants. No difference in detoxifying enzyme activity was detected.

The antennae of Grapholita molesta (Busck) are more susceptible to sex pheromone-induced sensory adaptation than the antennae of Choristoneura rosaceana (Harris). Adaptation is detectable in G. molesta at 1/1,000th the aerial concentration of pheromone (i.e., 5.0 x 10(-7) ng Z8-12:OAc/ml air) that is required to induce detectable adaptation in C. rosaceana (i.e., 5.0 x 10(-4) ng Z11-14:OAc/ml air). In addition, the predicted concentration of pheromone required to induce 50% adaptation in G. molesta (i.e., 1.2 x 10(-3) ng Z8-12:OAc/ml air) after 15 min of exposure is only one sixth the estimated aerial concentration required to induce the same level of adaptation in C. rosaceana (i.e., 7.5 x 10(-3) ng Z11-14:OAc/ml air) after 15 min of exposure. Sixteen percent and 28% adaptation of G. molesta antennae is predicted after 15 and 30 min of exposure to the equivalent of 1 ng Z8-12:OAc/m(3) air (i.e., 1 x 10(-6) ng Z8-12:OAc/ml air). The predicted level of adaptation in C. rosaceana antennae, however, is only 1.5 and 9.9% after 15 and 30 min of exposure, respectively, to the equivalent of 1 ng Z11-14:OAc/m(3) air (i.e., 1 x 10(-6) ng Z11-14:OAc/ml air). The approximately three-fold greater level of sensory adaptation in G. molesta antennae after 30 min of exposure to a pheromone concentration measured in pheromone-treated orchards (i.e., 1 ng/m(3)) may be one reason why this species is more readily controlled than C. rosaceana using mating disruption.

A new nylon membrane bag (NMB) assay was developed for studies to determine the effect of chemicals added to soil on survival of soilborne plant pathogens. The rapid and effective assay can be used to study organisms for which there are no selective media or for which a selective medium is expensive or difficult to prepare. This assay consists of placing pathogens inside a bag made of small-pore (0.22-μm) nylon filtration membrane, which is placed in soil and later retrieved to determine survival of the pathogens on nonselective media. Chemicals but not other microorganisms can enter the bag from the soil. Using this assay, Streptomyces scabies, Fusarium oxysporum f. sp. lycopersici race 3, and Ralstonia solanacearum were successfully recovered from soil after 72 h as demonstrated by growth on a semiselective Streptomyces medium (S. scabies) or nonselective potato dextrose agar medium (F. oxysporum f. sp. lycopersici race 3 and R. solanacearum) with minimal microbial contamination. Addition of acetic acid (200 mM) to soil killed 100% of S. scabies. SPK (a mixture of organic chemicals) at a concentration of 1,500 mg kg -1 of soil killed 83.3% of F. oxysporum f. sp. lycopersici race 3 culture plugs, 100% of F. oxysporum f. sp. lycopersici race 3 spores, and 97.2% of R. solanacearum cells. SPK at 1,000 mg kg -1 of soil killed 50% of F. oxysporum f. sp. lycopersici race 3 culture plugs, 68.2% of F. oxysporum f. sp. lycopersici race 3 spores, and 12% of R. solanacearum. Benlate (500 to 1,500 mg kg -1 of soil) did not kill the culture plugs of F. oxysporum f. sp. lycopersici race 3 but reduced the growth rate of F. oxysporum f. sp. lycopersici race 3. Benlate (500, 1,000, and 1,500 mg kg -1 of soil) reduced F. oxysporum f. sp. lycopersici race 3 spore germination by 39.4, 49.3, and 50.4%, respectively. Streptomycin sulfate (1,500, 800, 400, and 200 mg kg -1 of soil) caused 75.3, 21, 11.9, and 0.9% mortality, respectively, of R. solanacearum.

Nuclear pore complexes (NPCs) are vital to nuclear-cytoplasmic communication in eukaryotes. The yeast NPC-associated TREX-2 complex, also known as the Thp1-Sac3-Cdc31-Sus1 complex, is anchored on the NPC via the nucleoporin Nup1, and is essential for mRNA export. Here we report the identification and characterization of the putative Arabidopsis thaliana TREX-2 complex and its anchoring nucleoporin. Physical and functional evidence support the identification of the Arabidopsis orthologs of yeast Thp1 and Nup1. Of three Arabidopsis homologs of yeast Sac3, two are putative TREX-2 components, but, surprisingly, none are required for mRNA export as they are in yeast. Physical association of the two Cdc31 homologs, but not the Sus1 homolog, with the TREX-2 complex was observed. In addition to identification of these TREX-2 components, direct interactions of the Arabidopsis homolog of DSS1, which is an established proteasome component in yeast and animals, with both the TREX-2 complex and the proteasome were observed. This suggests the possibility of a link between the two complexes. Thus this work has identified the putative Arabidopsis TREX-2 complex and provides a foundation for future studies of nuclear export in Arabidopsis.

The viral genome-linked protein, VPg, of potyviruses is a multifunctional protein involved in viral genome translation and replication. Previous studies have shown that both eukaryotic translation initiation factor 4E (eIF4E) and eIF4G or their respective isoforms from the eIF4F complex, which modulates the initiation of protein translation, selectively interact with VPg and are required for potyvirus infection. Here, we report the identification of two DEAD-box RNA helicase-like proteins, PpDDXL and AtRH8 from peach (Prunus persica) and Arabidopsis (Arabidopsis thaliana), respectively, both interacting with VPg. We show that AtRH8 is dispensable for plant growth and development but necessary for potyvirus infection. In potyvirus-infected Nicotiana benthamiana leaf tissues, AtRH8 colocalizes with the chloroplast-bound virus accumulation vesicles, suggesting a possible role of AtRH8 in viral genome translation and replication. Deletion analyses of AtRH8 have identified the VPg-binding region. Comparison of this region and the corresponding region of PpDDXL suggests that they are highly conserved and share the same secondary structure. Moreover, overexpression of the VPg-binding region from either AtRH8 or PpDDXL suppresses potyvirus accumulation in infected N. benthamiana leaf tissues. Taken together, these data demonstrate that AtRH8, interacting with VPg, is a host factor required for the potyvirus infection process and that both AtRH8 and PpDDXL may be manipulated for the development of genetic resistance against potyvirus infections.

The replication of positive-strand RNA viruses occurs in cytoplasmic membrane-bound virus replication complexes (VRCs). Depending on the virus, distinct cellular organelles such as the endoplasmic reticulum (ER), chloroplast, mitochondrion, endosome, and peroxisome are recruited for the formation of VRC-associated membranous structures. Previously, the 6,000-molecular-weight protein (6K) of plant potyviruses was shown to be an integral membrane protein that induces the formation of 6K-containing membranous vesicles at endoplasmic reticulum (ER) exit sites for potyvirus genome replication. Here, we present evidence that the 6K-induced vesicles predominantly target chloroplasts, where they amalgamate and induce chloroplast membrane invaginations. The vesicular transport pathway and actomyosin motility system are involved in the trafficking of the 6K vesicles from the ER to chloroplasts. Viral RNA, double-stranded RNA, and viral replicase components are concentrated at the 6K vesicles that associate with chloroplasts in infected cells, suggesting that these chloroplast-bound 6K vesicles are the site for potyvirus replication. Taken together, these results suggest that plant potyviruses sequentially recruit the ER and chloroplasts for their genome replication.

Basic helix-loop-helix (bHLH) proteins constitute a large family of transcriptional regulators in plants. Although they have been shown to play important roles in a wide variety of developmental processes, relatively few have been functionally characterized. Here, we describe the map-based cloning of the Lotus japonicus ROOTHAIRLESS1 (LjRHL1) locus. Deleterious mutations in this locus prevent root hair development, which also aborts root hair-dependent colonization of the host root by nitrogen-fixing bacteria. We show that the LjRHL1 gene encodes a presumed bHLH transcription factor that functions in a nonredundant manner to control root hair development in L. japonicus. Homology search and cross-species complementation experiments defined three members of the Arabidopsis (Arabidopsis thaliana) bHLH protein family, At2g24260, At4g30980, and At5g58010, as functionally equivalent to LjRHL1. Curiously, At2g24260 and At4g30980 mRNA species accumulate independently from the known positive regulators of root hair cell fate, while all three genes act in a partially redundant manner to regulate root hair development in Arabidopsis.

Fish emulsion (FE) added to a sandy-loam soil at 1 and 2% rates reduced the viability of Verticillium dahliae microsclerotia by 39 and 74% in 1 day, 87 and 98% in 3 days, and 95 and 99% in 6 days, respectively. The immediate kill of microsclerotia indicated that FE contains toxic substances. We found in FE high concentrations (400 mmol/liter) of organic acids, including some known toxicants. Glycolic, acetic, formic, n-butyric, and propionic acids were the major organic acids detected in FE at the proportions of 52.5, 26.9, 7.9, 7.2, and 4.7%, respectively. In solution assays, the viability of V. dahliae microsclerotia treated for 24 h in 1, 2, 5, and 10% FE (pH 3.6 to 3.0) or a mixture of organic acids (pH 4.1 to 3.9) equivalent to the proportions in FE was reduced by 74, 94, 97, and 99% or 81, 91, 98, and 99%, respectively. The viability of microsclerotia was increased when the treatment solutions were buffered to pH 6.0. The organic acids mixtures and formic (0.025%) and acetic (0.1%) acids were toxic to Pythium ultimum. A mixture of organic acids (1, 2, and 4%) provided immediate protection of cucumber seedlings from damping-off in P. ultimum-infested muck and sandy-loam soils but not in peat-based mix. FE (1 and 2%) provided immediate protection of cucumber seedlings from damping-off in an infested muck soil, and disease protection was consistent when planting was delayed for 7, 14, and 28 days after adding FE. FE (1, 2, and 4%) did not provide immediate protection of cucumber seedlings from damping-off in a P. ultimum-infested peat-based mix; however, disease suppression was evident when planting was delayed for 7, 14, and 21 days after adding FE. Real-time polymerase chain reaction analyses of the peat-based mix indicated that the P. ultimum populations in the FE-amended mix declined over time. This study suggests that these organic acids in FE played a major role in pathogen or disease suppression, depending on the soil and substrate.

Single-stranded positive-sense RNA viruses induce the biogenesis of cytoplasmic membranous vesicles, where viral replication takes place. However, the mechanism underlying this characteristic vesicular proliferation remains poorly understood. Previously, a 6-kDa potyvirus membrane protein (6K) was shown to interact with the endoplasmic reticulum (ER) and to induce the formation of the membranous vesicles. In this study, the involvement of the early secretory pathway in the formation of the 6K-induced vesicles was investigated in planta. By means of live-cell imaging, it was found that the 6K protein was predominantly colocalized with Sar1, Sec23, and Sec24, which are known markers of ER exit sites (ERES). The localization of 6K at ERES was prevented by the coexpression of a dominant-negative mutant of Sar1 that disables the COPII activity or by the coexpression of a mutant of Arf1 that disrupts the COPI complex. The secretion of a soluble secretory marker targeting the apoplast was arrested at the level of the ER in cells overexpressing 6K or infected by a potyvirus. This blockage of protein trafficking out of the ER by 6K and the distribution of 6K toward the ERES may account for the aggregation of the 6K-bound vesicles. Finally, virus infection was reduced when the accumulation of 6K at ERES was inhibited by impairing either the COPI or COPII complex. Taken together, these results imply that the cellular COPI and COPII coating machineries are involved in the biogenesis of the potyvirus 6K vesicles at the ERES for viral-genome replication.

A naturally occurring Rsv4 resistance-breaking isolate (L-RB) and a closely related non-resistance-breaking isolate (L) of Soybean mosaic virus (SMV) were identified in soybean fields in London, Ontario, Canada. The viral genomes of L and L-RB were completely sequenced. Each isolate has a 9585-nucleotide genome with a single open reading frame encoding a polyprotein of approximately 350 kDa. L-RB and L have a very high sequence similarity (99.6%) at both the nucleotide and amino acid levels. Phylogenetic analysis showed that the two isolates belong to the G2 pathotype. Pathogenicity predictions of all virus/soybean combinations, based on the phylogenetic profile, were confirmed by pathogenicity tests using L and L-RB isolates and soybeans carrying different resistance genes, with an exception that L-RB infected a soybean cultivar carrying Rsv4 resistance. The temporal and spatial proximity of L and L-RB and their high sequence similarity suggest L-RB was likely derived from the SMV-L quasispecies. Recombination analysis did not reveal the evidence of genetic recombination for the emergence of L-RB. Mutations introduced by virus-encoded RNA-dependent RNA polymerase during viral genome replication and selection pressure probably contributed to the occurrence of L-RB.

RNA recombination is one of the two major factors that create RNA genome variability. Assessing its incidence in plant RNA viruses helps understand the formation of new isolates and evaluate the effectiveness of crop protection strategies. To search for recombination in Soybean mosaic virus (SMV), the causal agent of a worldwide seed-borne, aphid-transmitted viral soybean disease, we obtained all full-length genome sequences of SMV as well as partial sequences encoding the N-terminal most (P1 protease) and the C-terminal most (capsid protein; CP) viral protein. The sequences were analyzed for possible recombination events using a variety of automatic and manual recombination detection and verification approaches. Automatic scanning identified 3, 10, and 17 recombination sites in the P1, CP, and full-length sequences, respectively. Manual analyses confirmed 10 recombination sites in three full-length SMV sequences. To our knowledge, this is the first report of recombination between distinct SMV pathotypes. These data imply that different SMV pathotypes can simultaneously infect a host cell and exchange genetic materials through recombination. The high incidence of SMV recombination suggests that recombination plays an important role in SMV evolution. Obtaining additional full-length sequences will help elucidate this role.

Gregor Mendel's study of the common garden pea (Pisum sativum) provided the fundamentals for modern genetics and plant breeding and highlighted the utility and value of model organisms. One hundred and forty-three years later, insight into the genome structure of a model legume, Lotus japonicus, might provide the key to sustainable agriculture.

The contents of sulfur amino acids in seeds of common bean ( Phaseolus vulgaris L.) are suboptimal for nutrition. They accumulate large amounts of a gamma-glutamyl dipeptide of S-methyl-cysteine, a nonprotein amino acid that cannot substitute for methionine or cysteine in the diet. Protein accumulation and amino acid composition were characterized in three genetically related lines integrating a progressive deficiency in major seed storage proteins, phaseolin, phytohemagglutinin, and arcelin. Nitrogen, carbon, and sulfur contents were comparable among the three lines. The contents of S-methyl-cysteine and gamma-glutamyl-S-methyl-cysteine were progressively reduced in the mutants. Sulfur was shifted predominantly to the protein cysteine pool, while total methionine was only slightly elevated. Methionine and cystine contents (mg per g protein) were increased by up to ca. 40%, to levels slightly above FAO guidelines on amino acid requirements for human nutrition. These findings may be useful to improve the nutritional quality of common bean.

Background: Virus infection induces the activation and suppression of global gene expression in the host. Profiling gene expression changes in the host may provide insights into the molecular mechanisms that underlie host physiological and phenotypic responses to virus infection. In this study, the Arabidopsis Affymetrix ATH1 array was used to assess global gene expression changes in Arabidopsis thaliana plants infected with Plum pox virus (PPV). To identify early genes in response to PPV infection, an Arabidopsis synchronized single-cell transformation system was developed. Arabidopsis protoplasts were transfected with a PPV infectious clone and global gene expression changes in the transfected protoplasts were profiled., Results: Microarray analysis of PPV-infected Arabidopsis leaf tissues identified 2013 and 1457 genes that were significantly (Q < or = 0.05) up- (> or = 2.5 fold) and downregulated (< or = -2.5 fold), respectively. Genes associated with soluble sugar, starch and amino acid, intracellular membrane/membrane-bound organelles, chloroplast, and protein fate were upregulated, while genes related to development/storage proteins, protein synthesis and translation, and cell wall-associated components were downregulated. These gene expression changes were associated with PPV infection and symptom development. Further transcriptional profiling of protoplasts transfected with a PPV infectious clone revealed the upregulation of defence and cellular signalling genes as early as 6 hours post transfection. A cross sequence comparison analysis of genes differentially regulated by PPV-infected Arabidopsis leaves against uniEST sequences derived from PPV-infected leaves of Prunus persica, a natural host of PPV, identified orthologs related to defence, metabolism and protein synthesis. The cross comparison of genes differentially regulated by PPV infection and by the infections of other positive sense RNA viruses revealed a common set of 416 genes. These identified genes, particularly the early responsive genes, may be critical in virus infection., Conclusion: Gene expression changes in PPV-infected Arabidopsis are the molecular basis of stress and defence-like responses, PPV pathogenesis and symptom development. The differentially regulated genes, particularly the early responsive genes, and a common set of genes regulated by infections of PPV and other positive sense RNA viruses identified in this study are candidates suitable for further functional characterization to shed lights on molecular virus-host interactions.

Confined livestock production farms typically store their wastes prior to land application. Here, we employed three complementary approaches to evaluate changes in the population structure and stability of virulence genes in Escherichia coli during manure storage on a commercial farm that housed healthy swine. Isolates were genotyped by repetitive extragenic palindromic PCR using the BOXA1R primer and evaluated for the presence of selected virulence genes by PCR. Isolates obtained from the manure holding tank (n = 392) carried estB, fedA, stx(2e), astA, paa, aida-I, and sepA at lower frequencies than isolates obtained from fresh feces (n = 412). Fresh fecal material from the barn was added into diffusion chambers and immersed in the manure holding tank for 7 weeks. The fecal E. coli population was initially dominated by a single genotype, all isolates of which carried fedA and aida-I. After 7 weeks, a genotype that did not carry any virulence genes dominated the surviving population. In a second experiment, 48 fecal isolates of E. coli that varied in their genotypes and virulence gene complement were incubated in diffusion chambers in the manure holding tank for 3 weeks. Over 95% of the inoculum population carried at least one virulence gene, whereas after 3 weeks 90% of the recovered isolates carried no virulence genes. Taken together, these results indicate that during commercial manure storage, there was a significant reduction in the carriage of these virulence genes by E. coli. We propose that loss of virulence genes from enteric pathogens in the farm and in natural environments may, if generalized, contribute to the attenuation of a public health risk from contamination with agricultural wastes.

Synthesis and accumulation of seed storage proteins (SSPs) is an important aspect of the seed maturation program. Genes encoding SSPs are specifically and highly expressed in the seed during maturation. However, the mechanisms that repress the expression of these genes in leaf tissue are not well understood. To gain insight into the repression mechanisms, we performed a genetic screen for mutants that express SSPs in leaves. Here, we show that mutations affecting BRAHMA (BRM), a SNF2 chromatin-remodeling ATPase, cause ectopic expression of a subset of SSPs and other embryogenesis-related genes in leaf tissue. Consistent with the notion that such SNF2-like ATPases form protein complexes in vivo, we observed similar phenotypes for mutations of AtSWI3C, a BRM-interacting partner, and BSH, a SNF5 homolog and essential SWI/SNF subunit. Chromatin immunoprecipitation experiments show that BRM is recruited to the promoters of a number of embryogenesis genes in wild-type leaves, including the 2S genes, expressed in brm leaves. Consistent with its role in nucleosome remodeling, BRM appears to affect the chromatin structure of the At2S2 promoter. Thus, the BRM-containing chromatin-remodeling ATPase complex involved in many aspects of plant development mediates the repression of SSPs in leaf tissue.

The relative attractiveness of synthetic Choristoneura rosaceana (Harris) pheromone consisting of the major compound Z-11-tetradecenyl acetate (Z11-14:OAc), or the major compound plus one, two, or all three of the minor compounds E-11-tetradecenyl acetate (E11-14:OAc), Z-11-tetradecenol (Z11-14:OH), and Z-11-tetradecenal (Z11-14:Ald), in amounts similar to those in the natural pheromone, was compared in a flight tunnel and in apple orchards. In the flight tunnel, there was an increase in the proportion of moths that were activated when E11-14:OAc was combined with the main compound, but no detectible additional increases in response with the addition of Z11-14:OH and Z11-14:Ald. The time required for activation was twice as long when using the major compound than when using the major compound and one or more of the minor compounds. There was a trend of increase in the proportion of males initiating the take-off, lock-on, close-in, and touchdown phases of upwind flight when progressively more complete blends were used. The time required for the initiation of these behavioral phases was statistically similar for each treatment. The proportion of moths landing at the pheromone source was 10 times greater when using the complete blend than when using the main compound alone. Similar results were obtained in apple orchards, where the four-compound blend was 17-55 times more attractive than the main compound. The superior attractiveness of the four-compound pheromone compared with the major compound, or to the major compound plus the minor compound E11-14:OAc, suggests that it would be the most effective synthetic pheromone if competitive attraction were an important mechanism of disruption in this species.

Compatible virus infection induces and suppresses host gene expression at the global level. These gene-expression changes are the molecular basis of symptom development and general stress and defence-like responses of the host. To assess transcriptional changes in soybean plants infected with soybean mosaic virus (SMV), the first soybean trifoliate leaf, immediately above the SMV-inoculated unifoliate leaf, was sampled at 7, 14 and 21 days post-inoculation (p.i.) and subjected to microarray analysis. The identified changes in gene expression in soybean leaves with SMV infection at different time points were associated with the observed symptom development. By using stringent selection criteria (>or=2- or

The regulation of ACC synthase (ACS) genes was studied in early ('Early Golden') and late ('Shiro') Japanese plum cultivars (Prunus salicina L.) in order to determine the role of this gene family in fruit ripening. Of the four Ps-ACS cDNAs isolated, two (Ps-ACS1 and -3) showed differential expression between the two cultivars. Ps-ACS1 accumulated during fruit ripening of 'Early Golden' ('EG') and 'Shiro' ('SH') in ethylene-dependent and -independent manners, respectively. Ps-ACS3a transcripts accumulated throughout fruit development and during 'EG' fruit ripening. Ps-ACS3b was detected only during ripening of 'SH' fruit. Furthermore, Ps-ACS3a transcript accumulation was negatively regulated by ethylene, whereas Ps-ACS3b was positively induced by the hormone. In both cultivars, the expression of Ps-ACS4 and -5 is under positive and negative feedback control by ethylene, respectively. Genetic analyses of 'EG' and 'SH' cultivars demonstrated that 'EG' is homozygous for Ps-ACS3a whereas 'SH' is heterozygous for Ps-ACS3 (a/b). The role of ethylene-overproducer 1-like in delaying fruit ripening by interacting with Ps-ACS proteins was also studied. The effect of the plant hormones, auxin, gibberellin, and cytokinin, in regulating ethylene production by promoting the induction of the different Ps-ACS mRNAs in plum was investigated. A model is presented in which differences in Ps-ACS alleles and gene expression between early and late plums are critical in determining the ripening behaviour of the cultivars.

The efficacy of 3M Sprayable Pheromone for Grape Berry Moth, BASF RAK 1R pheromone dispensers, Isomate GBM pheromone dispensers, and an insecticide program were compared during two seasons for controlling grape berry moth, Endopiza viteana (Clemens), in Niagara peninsula, Ontario, Canada, vineyards. The average number of E. viteana captured in pheromone-baited traps in plots treated with BASF RAK 1R and Isomate GBM dispensers was reduced by 90-100% compared with the average number captured in insecticide-treated plots, indicating a high level of mating disruption. By contrast, there was no difference in the number of moths captured in plots treated with sprayable pheromone and in plots treated with insecticide during the second flight of 1999, suggesting that the sprayable pheromone did not affect the mate-seeking ability of male moths. During the third flight of 1999, and the three flights of 2000, however, the average number of E. viteana captured in plots treated with sprayable pheromone was reduced by 50-99% compared with the average number captured in insecticide-treated blocks, indicating a moderate-to-high level of mating disruption. There was no detectable difference in feeding injury to grape clusters when E. viteana was controlled using two application rates of a sprayable pheromone formulation, two hand-applied pheromone dispensers, or a conventional, insecticide-control program. The disparity between estimated disruption and crop damage in plots treated with sprayable pheromone during the second flight of 1999 suggests that pheromone-baited traps may not provide a reliable estimate of the level of mating disruption when using sprayable pheromone.

Two free-living nitrogen-fixing bacterial strains, N6 and N7(T), were isolated from corn rhizosphere. A polyphasic taxonomic approach, including morphological characterization, Biolog analysis, DNA-DNA hybridization, and 16S rRNA, cpn60 and nifH gene sequence analysis, was taken to analyse the two strains. 16S rRNA gene sequence analysis indicated that strains N6 and N7(T) both belonged to the genus Azospirillum and were closely related to Azospirillum oryzae (98.7 and 98.8 % similarity, respectively) and Azospirillum lipoferum (97.5 and 97.6 % similarity, respectively). DNA-DNA hybridization of strains N6 and N7(T) showed reassociation values of 48 and 37 %, respectively, with A. oryzae and 43 % with A. lipoferum. Sequences of the nifH and cpn60 genes of both strains showed 99 and approximately 95 % similarity, respectively, with those of A. oryzae. Chemotaxonomic characteristics (Q-10 as quinone system, 18 : 1omega7c as major fatty acid) and G+C content of the DNA (67.6 mol%) were also similar to those of members of the genus Azospirillum. Gene sequences and Biolog and fatty acid analysis showed that strains N6 and N7(T) differed from the closely related species A. lipoferum and A. oryzae. On the basis of these results, it is proposed that these nitrogen-fixing strains represent a novel species. The name Azospirillum zeae sp. nov. is suggested, with N7(T) (=NCCB 100147(T)=LMG 23989(T)) as the type strain.

We have cloned two new lepidopteran octopamine transporters (OATs), members of the solute-linked carrier family 6 (SLC6) of nutrient transporters, from the CNS of the European corn borer Ostrinia nubilalis and the cabbage white Pieris rapae. Comparison of these sequences with the previously cloned OAT from the cabbage looper Trichoplusia ni showed that the T. ni OAT sequence previously reported was truncated by 74 amino acids at the N-terminus. The cytoplasmic N-termini deduced here are considerably longer than the N-termini of other monoamine transporters in the SLC6 family and contain many more high-probability serine- and threonine-phosphorylation sites. Monoamine uptake and competitive inhibition studies on baculovirus-infected Sf9 cells expressing these three cloned OATs indicate that they are able to transport tyramine, octopamine and dopamine with high affinity (K(m) and K(i) range, 0.4 microM-2.7 microM) and capacity ((3)H-dopamine uptake by TrnOAT, 2.5 pmol/well/min). We aimed to examine the role of the N-terminus of OAT by comparing the properties of the full-length T. ni OAT with those of the previously reported N-truncated version. Results for the new full-length T. ni OAT showed no difference in the protein's affinity for octopamine or dopamine, although at low levels of viral infection it did show slightly higher transport activity ((3)H-dopamine uptake by truncated TrnOAT, 1.5 pmol/well/min). Treatment of Sf9 cells expressing full-length or truncated TrnOAT with a variety of protein kinase activators and inhibitors, however, did not change transporter activity. Neither an intact N-terminus, nor apparently a particular phosphorylation state of this extended N-terminus, is required for OAT to transport monoamines.

A micropropagation approach was developed for nine ornamental Prunus species, P. americana, P. cistena, P. glandulosa, P. serrulata 'Kwanzan', P. laurocerasus, P. sargentii, P. tomentosa, P. triloba, P. virginiana 'Schubert', commercially important in North America, and GF305 peach, commonly used for Prunus virus indexing. The micropropagation cycle based on proliferation of vegetative tissues includes establishment of tissue culture through introduction of shoot meristems in vitro, shoot proliferation, root induction and plant acclimatization steps and can be completed in 5 months. A meristem sterilization protocol minimized bacterial and fungal contamination. Multiple shoot formation in ornamental Prunus was obtained through the use of 1 mg l(-1) 6-benzyladenine. For GF305 peach, alteration in the sugar composition, fructose instead of sucrose, and addition of 1 mg l(-1 )ferulic acid had a significant impact on the shoot proliferation rate and maintenance of long-term in vitro culture. Rooting and plant acclimatization conditions were improved using a two-step protocol with a 4-day root induction in indole-3-butiric acid (IBA)-containing media with consequent 3-week root elongation in IBA-free media. One-month incubation of rooted shoots in a vermiculite-based medium resulted in additional shoot and root growth and provided better acclimatization and plant recovery. The micropropagation approach can be used for maintenance of the clonal properties for Prunus spp. as well as a protocol to support meristem therapy against viral infection.

Mammalian sulfotransferases (EC 2.8.2) are involved in many important facets of steroid hormone activity and metabolism. In this study, Arabidopsis AtST4a and AtST1 were identified and characterized as brassinosteroid sulfotransferases that appear to be involved in different aspects of hormone regulation. The two proteins share 44% identity in amino acid sequence, and belong to different plant sulfotransferase families. AtST4a was specific for biologically active end products of the brassinosteroid pathway. The enzyme sulfated brassinosteroids with diverse side-chain structures, including 24-epibrassinosteroids and the naturally occurring (22R, 23R)-28-homobrassinosteroids. AtST4a belongs to a small subfamily of sulfotransferases having two other members, AtST4b and -c. Among the three recombinant enzymes, only AtST4a was catalytically active with brassinosteroids. Transcript expression of AtST4 subfamily members was largely specific to the root. AtST4b- and -c transcript levels were induced by treatment with trans-zeatin, while AtST4a was repressed under the same conditions, supporting a divergent function of AtST4a. Co-regulation of AtST4b and -c correlated with their location in tandem on chromosome 1. AtST1 was stereospecific for 24-epibrassinosteroids, with a substrate preference for the metabolic precursor 24-epicathasterone, and exhibited catalytic activity with hydroxysteroids and estrogens. To gain more insight into this dual activity with plant and mammalian steroids, enzymatic activities of human steroid sulfotransferases toward brassinosteroids were characterized. The dehydroepiandrosterone sulfotransferase SULT2A1 displayed catalytic activity with a selected set of 24-epibrassinolide precursors, including 24-epicathasterone, with specific activities comparable to that measured for the endogenous substrate dehydroepiandrosterone. The comparable activity profiles of AtST1 and SULT2A1 suggest a similar architecture of the acceptor-binding site between the two enzymes, and may potentially reflect a common ability to conjugate certain xenobiotics.

A free-living diazotrophic strain, DS2(T), was isolated from corn rhizosphere. Polyphasic taxonomy was performed including morphological characterization, Biolog analysis, and 16S rRNA, cpn60 and nifH gene sequence analyses. 16S rRNA gene sequence analysis indicated that strain DS2(T) was closely related to the genus Azospirillum (96 % similarity). Chemotaxonomic characteristics (DNA G+C content 67.9 mol%; Q-10 quinone system; major fatty acid 18 : 1omega7c) were also similar to those of the genus Azospirillum. In all the analyses, including phenotypic characterization using Biolog analysis and comparison of cellular fatty acids, this isolate was found to be different from the closely related species Azospirillum lipoferum, Azospirillum oryzae and Azospirillum brasilense. On the basis of these results, a novel species is proposed for this nitrogen-fixing strain. The name Azospirillum canadense sp. nov. is suggested with the type strain DS2(T) (=NCCB 100108(T)=LMG 23617(T)).

The quality of water draining fields fertilized with liquid swine (Sus scrofa) manure (LSM) sidedressed into standing corn (Zea mays L.) at rates ranging from 0 to 94 m(3) ha(-1), either topdressed (TD) onto the surface, or injected (INJ) into the soil once annually for each of three consecutive years was evaluated. Liquid swine manure application rate was a critical driver of preferential flow of LSM to tile as detected by turbidity, concentrations of NH(4)(+)-N, dissolved reactive phosphorus (DRP), and the presence of enteric bacteria (Escherichia coli). Contaminant movement to drains occurred immediately after 75 and 94 m(3) LSM ha(-1) were injected (e.g., 2.5 mg DRP L(-1), 3-yr average). With injection of 56 m(3) ha(-1) or less, drainage water was not turbid and concentrations of NH(4)(+)-N, DRP, and enteric bacteria were dramatically lower than with the higher rates, even when tiles flowed freely during manure application. Application method also affected tile water quality. With TD applications (37 and 56 m(3) ha(-1)), nutrients and bacteria did not move to tiles at the time of application, but with rains that fell within 3 d after application, concentrations increased (e.g., 0.1 mg DRP L(-1)), although less than with INJ. Overall, sidedress injection rates that supplied adequate crop nutrients did not compromise drainage water quality.

In order to minimize odor and manage nutrients in liquid pig manure we need to be able to predict what operational practices most influence the concentrations of volatile fatty acids (VFAs), ammonium nitrogen (NH(4)(+)-N), and other nutrients present in the manure. To determine this, we collected manure from 15 pig operations in southwestern Ontario in the fall of 2001 and 2002 and spring of 2002 and 2003. The manure was stored in concrete tanks at all operations. Manure from finishing pigs had the highest concentration of VFAs, NH(4)(+)-N, and other nutrients, followed by manure from mixed operations, and then manure from sow operations. The average concentration of total VFAs and NH(4)(+)-N in finishing pig manure was 166 mM compared with 36 and 99 mM, respectively, in sow manure. Total N, P, and K were 2.3, 2.5, and 1.7 times greater, respectively, in finishing pig compared with sow manure. There was no seasonal or year to year variation in amount. The diet of the pigs, use of feed additives or antibiotics, location of tanks, and whether the tanks were covered or mixed were not significant factors contributing to the difference in manure chemistry. The main reason for the differences between the three types of manure was manure dilution. The average dry matter content of finishing pig manure was 4.5 times that of sow manure. This was due to larger density of pigs in finishing compared with sow operations, less manure storage capacity per pig for finishing compared with sow operations, and more wash water being used for sow operations.

In legumes, Nod-factor signaling by rhizobia initiates the development of the nitrogen-fixing nodule symbiosis, but the direct cell division stimulus that brings about nodule primordia inception in the root cortex remains obscure. We showed that Lotus japonicus plants homozygous for a mutation in the HYPERINFECTED 1 (HIT1) locus exhibit abundant infection-thread formation but fail to initiate timely cortical cell divisions in response to rhizobial signaling. We demonstrated that the corresponding gene encodes a cytokinin receptor that is required for the activation of the nodule inception regulator Nin and nodule organogenesis.

Inflammatory bowel disease (IBD) represents a spectrum of diseases in which inflammation leads to acute and chronic gut injury. It is a growing health issue for which no cure exists. The pathogenesis is multifactorial with links to infectious and environmental events that trigger disease in genetically predisposed individuals. Treatment of the two major forms of IBD, Crohn's disease and ulcerative colitis, involves the reduction of inflammation with toxic immunosuppressive drugs or blocking of the pro-inflammatory effects of tumour necrosis factor-alpha (TNF-alpha) with antibodies. Here, we show that the oral administration of transgenic low-alkaloid tobacco expressing the contra-inflammatory cytokine human interleukin-10 (hIL-10) reduces the severity of colitis by down-regulating TNF-alpha expression directly at the sites of inflammation in IBD-susceptible IL-10(-/-) mice. hIL-10 expressed in plants is biologically active and displays resistance to gastrointestinal degradation. Dietary supplementation with plant tissue delivering up to 9 microg of hIL-10 daily for 4 weeks was well tolerated by treated mice. Gut histology was significantly improved relative to controls (P = 0.002), and was correlated with a decrease in small bowel TNF-alpha mRNA levels and an increase in IL-2 and IL-1beta mRNA levels. Transgenic plants expressing IL-10 to directly attenuate TNF-alpha expression at sites of inflammation in the gut may become a useful new approach in the luminal therapy of IBD.

A new species of the anamorphic genus Rhexocercosporidium is described. Isolates of a Rhex-Rhexocercosporidium sp. were obtained from ginseng (Panax quinquefolius) roots with symptoms of rusted root. These isolates were found to be genetically and morphologically distinct from the only described species in this genus, R. carotae. Sequence data from the ribosomal DNA region spanning the internal transcribed spacers 1 and 2 and from a portion of the 3-tubulin gene of the ginseng Rhexocercosporidium were compared to those of R. carotae. Parsimony analyses of sequence data showed that R. carotae and the ginseng isolates belonged to distinct but closely related clades. Conidia of a typical ginseng isolate were significantly shorter and possessed fewer septa than R. carotae but shared rhexolytic secession of conidia with R. carotae. The binomial Rhexocercosporidium panacis is proposed to accommodate isolates of this genus that are associated with the rusted root disease.

Field trials were carried out to test the effect of phosphonate fungicide (AG3) on the severity of clubroot of bok choy (Brassica rapa var. chinensis) and cabbage (B. rapa var. perkinensis and B. oleracea var. capitata) in commercial Ontario muck fields with a clubroot history. Disease severity also was examined in the same infested soil under greenhouse and microplot conditions. In microplot trials with bok choy, AG3 phosphonate concentrations of 0.07 and 0.14% a.i. applied before or after planting consistently reduced clubroot severity (1-to-4 rating) by 0.8 to 1.6 when planted in May or June. However, only the 0.14% a.i. preplanting treatment was effective in trials in July and August. Postplanting drenches of 0.14% a.i. were consistently effective throughout the season. Fresh weight of bok choy was increased or not affected by phosphonate treatments. Under field conditions, one (0.07, 0.14, and 0.21% a.i.) or two (0.07% a.i.) postplant-ing drench applications of phosphonate significantly reduced the incidence of clubroot by 52 to 87% and severity by 1.7 to 2.5 on bok choy in 2004 but not in 2005. In the 2004 trial, two applications of 0.07% a.i. AG3 phosphonate reduced the severity of clubroot comparably to single applications at 0.14 and 0.21% a.i. rates. Fresh weight of bok choy was increased by 34 to 86% with all phosphonate drench treatments in both years. With cabbage, AG3 postplanting drench treatments consistently reduced the severity of clubroot (1-to-5 rating) by a range of 0.7 to 3.3 under greenhouse, microplot, and field conditions. In the greenhouse, a single drench application of 0.07 and 0.14% a.i. AG3 phosphonate 1 day after transplanting cabbage seedlings to the infested muck soil reduced clubroot severity by 2.6 to 3.3 and increased fresh weight of cabbage tops by 66 to 69%. Similar results were seen with cabbage trials under both microplot and field conditions at all AG3 postplanting drench concentrations.

Lotus japonicus har1 mutants respond to inoculation with Mesorhizobium loti by forming an excessive number of nodules due to genetic lesions in the HAR1 autoregulatory receptor kinase gene. In order to expand the repertoire of mutants available for the genetic dissection of the root nodule symbiosis (RNS), a screen for suppressors of the L. japonicus har1-1 hypernodulation phenotype was performed. Of 150,000 M2 plants analyzed, 61 stable L. japonicus double-mutant lines were isolated. In the context of the har1-1 mutation, 26 mutant lines were unable to form RNS, whereas the remaining 35 mutant lines carried more subtle symbiotic phenotypes, either forming white ineffective nodules or showing reduced nodulation capacity. When challenged with Glomus intraradices, 18 of the 61 suppressor lines were unable to establish a symbiosis with this arbuscular mycorrhiza fungus. Using a combined approach of genetic mapping, targeting induced local lesions in genomics, and sequencing, all non-nodulating mutant lines were characterized and shown to represent new alleles of at least nine independent symbiotic loci. The class of mutants with reduced nodulation capacity was of particular interest because some of them may specify novel plant functions that regulate nodule development in L. japonicus. To facilitate mapping of the latter class of mutants, an introgression line, in which the har1-1 allele was introduced into a polymorphic background of L. japonicus ecotype MG20, was constructed.

Acidic electrolyzed water (AEW), known to have germicidal activity, was obtained after electrolysis of 0.045% aqueous solution of sodium chloride. Freshly prepared AEW (pH 2.3-2.6, oxidation-reduction potential 1007-1025 mV, and free active chlorine concentration 27-35 ppm) was tested in vitro and (or) on tomato foliage and seed surfaces for its effects on the viability of plant pathogen propagules that could be potential seed contaminants. Foliar sprays of AEW were tested against bacterial spot disease of tomato under greenhouse and field conditions. The viability of propagules of Xanthomonas campestris pv. vesicatoria (bacterial spot pathogen), Streptomyces scabies (potato scab pathogen), and Fusarium oxysporum f.sp. lycopersici (root rot pathogen) was significantly reduced 4-8 log units within 2 min of exposure to AEW. Immersion of tomato seed from infected fruit in AEW for 1 and 3 min significantly reduced the populations of X. campestris pv. vesicatoria from the surface of the seed without affecting seed germination. Foliar sprays of AEW reduced X. campestris pv. vesicatoria populations and leaf spot severity on tomato foliage in the greenhouse. In the field, multiple sprays of AEW consistently reduced bacterial spot severity on tomato foliage. Disease incidence and severity was also reduced on fruit, but only in 2003. Fruit yield was either enhanced or not affected by the AEW sprays. These results indicate a potential use of AEW as a seed surface disinfectant or contact bactericide.

The development of novel strategies against plant viral diseases relies on a better understanding of molecular virus-host interactions. Here, we report an easy, efficient and reproducible protocol for Arabidopsis protoplast isolation and transfection to study the infection and replication of a potyvirus, Plum pox virus (PPV). Macerozyme and cellulose were used to release protoplasts from Arabidopsis leaf tissues, and polyethylene glycol-mediated DNA uptake was employed for transfection of a PPV infectious clone. Protoplast viability was monitored by fluorescein diacetate staining, and transfection efficiency was estimated by transient expression of the green fluorescent protein. The protocol allowed production of 95% viable mesophyll protoplasts and a successful transfection rate of 35%. The system was used further in a time-course experiment to investigate PPV viral RNA accumulation. It was found that 3 h post-transfection (hpt) in the transfected protoplasts viral RNA increased by about 150-fold and progressively accumulated to reach the maximum at 12 hpt. Viral RNA then decreased dramatically at 24 hpt reaching 40% of its peak level. Considering the availability of the whole genome microarrays, and other genomic resources of Arabidopsis, the synchronized single-cell (protoplast) infection system will be useful for elucidating early molecular events associated with PPV infection.

A glyoxalase I gene homologue (VdGLO1) was identified in the vascular wilt fungus Verticillium dahliae by sequence tag analysis of genes expressed during resting structure development. The results of the current study show that the gene encodes a putative 345 amino acid protein with high similarity to glyoxalase I, which produces S-D-lactoylglutathione from the toxic metabolic by-product methylglyoxal (MG). Disruption of the V. dahliae gene by Agrobacterium tumefaciens-mediated transformation resulted in enhanced sensitivity to MG. Mycelial growth of disruption mutants was severely reduced in the presence of 5 mmol/L MG. In contrast, spore production in liquid medium was abolished at 1 mmol/L MG, although not at physiologically relevant concentrations of

Six acetic acid producing, diazotrophic bacteria were isolated from soil adhering to corn roots. These isolates were shown to be Gluconacetobacter azotocaptans and they shared some features with G. johannae and G. diazotrophicus but differed on the basis of colony morphology on different media, use of carbon sources and use of l-amino acids as a nitrogen source. The species identity was confirmed using 16S rDNA sequence analysis, PCR amplification of 16S rRNA gene with species-specific primers and amplified rDNA restriction analysis. This is the first report of the presence of this bacteria on corn plants. Scope of the paper: This is the first report of the occurrence and association of Gluconacetobacter azotocaptans with corn.

Mutant lines of Lotus japonicus (Regel) Larsen that show defects in nodulation as well as in mycorrhiza formation are valuable resources for studying the events required for the establishment of functional symbioses. In this study, 11 mutant lines derived from a screen for genetic suppressors of har1-1 hypernodulation were assessed quantitatively for their ability to form arbuscular mycorrhizal (AM) symbiosis. The presence of extraradical mycelia, appressoria, intraradical hyphae, arbuscules and vesicles were scored. Roots of the har1-1 parental line were heavily colonised by six weeks after inoculation with the AM fungus Glomus intraradices showing the typical Arum-type colonisation pattern. Five mutants lacked internal root colonisation with blocks either at the surface of epidermal cells or at the outer tangential wall of cortical cells. These AM - lines showed some differences in relation to the amount of extraradical hyphae, the number of appressoria, and the degree of abnormal appressorium morphology. Four mutants had internal root colonisation but at a lower level than the parental line. Two mutants showed no difference from the parental line. Results of this study provide additional genetic resources for studying the mechanism of root colonisation by AM fungi.

L-asparaginases (EC 3.5.1.1) are hypothesized to play an important role in nitrogen supply to sink tissues, especially in legume-developing seeds. Two plant L-asparaginase subtypes were previously identified according to their K(+)-dependence for catalytic activity. An L-asparaginase homologous to Lupinus K(+)-independent enzymes with activity towards beta-aspartyl dipeptides, At5g08100, has been previously characterized as a member of the N-terminal nucleophile amidohydrolase superfamily in Arabidopsis. In this study, a K(+)-dependent L-asparaginase from Arabidopsis, At3g16150, is characterized. The recombinants At3g16150 and At5g08100 share a similar subunit structure and conserved autoproteolytic pentapeptide cleavage site, commencing with the catalytic Thr nucleophile, as determined by ESI-MS. The catalytic activity of At3g16150 was enhanced approximately tenfold in the presence of K(+). At3g16150 was strictly specific for L-Asn, and had no activity towards beta-aspartyl dipeptides. At3g16150 also had an approximately 80-fold higher catalytic efficiency with L-Asn relative to At5g08100. Among the beta-aspartyl dipeptides tested, At5g08100 had a preference for beta-aspartyl-His, with catalytic efficiency comparable to that with L-Asn. The phylogenetic analysis revealed that At3g16150 and At5g08100 belong to two distinct subfamilies. The transcript levels of At3g16150 and At5g08100 were highest in sink tissues, especially in flowers and siliques, early in development, as determined by quantitative RT-PCR. The overlapping spatial patterns of expression argue for a partially redundant function of the enzymes. However, the high catalytic efficiency suggests that the K(+)-dependent enzyme may metabolize L-Asn more efficiently under conditions of high metabolic demand for N.

The sweet steviol glycosides found in the leaves of Stevia rebaudiana Bert. are derived from the diterpene steviol which is produced from a branch of the gibberellic acid (GA) biosynthetic pathway. An understanding of the spatial organisation of the two pathways including subcellular compartmentation provides important insight for the metabolic engineering of steviol glycosides as well as other secondary metabolites in plants. The final step of GA biosynthesis, before the branch point for steviol production, is the formation of (-)-kaurenoic acid from (-)-kaurene, catalysed by kaurene oxidase (KO). Downstream of this, the first committed step in steviol glycoside synthesis is the hydroxylation of kaurenoic acid to form steviol which is then sequentially glucosylated by a series of UDP-glucosyltransferases (UGTs) to produce the variety of steviol glycosides. The subcellular location of KO and three of the UGTs involved in steviol glycoside biosynthesis was investigated by expression of GFP fusions and cell fractionation which revealed KO to be associated with the endoplasmic reticulum and the UGTs in the cytoplasm. It has also been shown by expressing the Stevia UGTs in Arabidopsis that the pathway can be partially reconstituted by recruitment of a native Arabidopsis glucosyltransferase.

Alcohol production from corn is gaining importance in Ontario, Canada, and elsewhere. A major cost of corn production is the cost of chemical fertilizers and these continue to increase in price. The competitiveness of alcohol with fossil fuels depends on access to low-cost corn that allows growers to earn a sustainable income. In this study we set out to determine if we can identify root-associated microorganisms from Ontario-grown corn that can enhance the nutrient flow to corn roots, directly or indirectly, and help minimize the use of extraneous fertilizer. Bacteria were isolated from corn rhizosphere and screened for their capacity to enhance corn growth. The bacteria were examined for their ability to fix nitrogen, solubilize phosphate, and produce indole acetic acid (IAA) and antifungal substances on potato dextrose agar. Bacterial suspensions were applied to pregerminated seed of four corn varieties (39D82, 39H84, 39M27, and 39T68) planted in sterilized sand and unsterilized cornfield soil. The plants were grown under greenhouse conditions for 30 days. Three isolates were identified as having growth-promoting effect. These bacteria were identified as to species by biochemical tests, fatty acid profiles, and 16S rDNA sequence analysis. Corn rhizosphere isolates, Gluconacetobacter azotocaptans DS1, Pseudomonas putida CQ179, and Azospirillum lipoferum N7, provided significant plant growth promotion expressed as increased root/shoot weight when compared to uninoculated plants, in sand and/or soil. All strains except P. putida CQ179 were capable of nitrogen fixation and IAA production. Azospirillum brasilense, however, produced significantly more IAA than the other isolates. Although several of the strains were also able to solubilize phosphate and produce metabolites inhibitory to various fungal pathogens, these properties are not considered as contributing to growth promotion under the conditions used in this study. These bacteria will undergo field tests for their effect on corn growth.

A formulation of phosphonate (AG3) was tested as a seed treatment for the control of Pythium damping-off of cucumber (Cucumis sativus L.) plants under controlled environment and field conditions. Cucumber seed were treated by soaking for 10 min in phosphonate solution. They were then planted into peat-based mix or sandy-loam soil mixed with Pythium aphanidermatum or P. ultimum inoculum or into muck soil naturally infested with P. irregulare, P. ultimum, and other Pythium spp. Under growth-room conditions, phosphonate seed treatment provided more than 80% control of damping-off in all infested substrates tested. Effective disease control was obtained even when treated seed were stored for 5 weeks and up to 18 months prior to planting. In microplots containing naturally infested muck soil, phosphonate seed treatment decreased the percentage of diseased cucumber plants and increased total fresh weights compared with untreated seed and phosphonate post-planting drench. In field-plot tests 6 weeks after planting treated seed in Pythium-infested muck soil, cucumber stands were 63% compared with 18% in the control, which had no phosphonate exposure, and 53% in the post-planting drench. Tests for potential phytotoxicity in the greenhouse showed that radish and bok choy germination was reduced by phosphonate treatment but corn, cucumber, soybean, sugar beet, tomato, and wheat were not affected. Phosphonate seed treatment is a cost-effective way of protecting cucumber plants from Pythium damping-off.

The wilt fungus Verticillium dahliae Kleb. produces desiccation- and cold-tolerant resting structures, known as microsclerotia, which are the primary source of disease inoculum in the field. In an exploration of the molecular mechanisms involved in the development of these important structures, we have identified in V. dahliae a differentially expressed, class II hydrophobin gene (VDH1). vdh1 mutants generated through targeted gene disruption show a severe reduction in microsclerotia production, indicating that the gene is important for this type of development. Although vdh1 mutants do produce normal conidiophores and spores, desiccation-tolerance of the spores is reduced. The VDH1 gene is not, however, needed for normal disease development in tomato. VDH1's functions are multi-faceted, and seem generally relevant to long-term survival in V. dahliae.