First Report of Soybean Vein Necrosis Disease Caused by Soybean vein necrosis-associated virus in Wisconsin and Iowa.

Several viral diseases of soybean (Glycine max) have been identified in the north-central U.S. soybean production area, which includes Wisconsin and Iowa (2). Previously, Soybean vein necrosis disease (SVND) caused by Soybean vein necrosis-associated virus was reported in Arkansas, Tennessee, and other southern states (4). In September 2012, soybean plants with symptoms similar to those reported for SVND (4) were observed in fields across Wisconsin and Iowa. Symptoms included leaf-vein and leaf chlorosis, followed by necrosis of the leaf veins and eventually necrosis of the entire leaf. Six samples with symptoms indicative of SVNaV were collected from research plots located at the West Madison Agricultural Research Station located in Madison, WI. An additional three samples were collected from three locations in central Iowa. Total RNA extracted from each sample using the Trizol Plus RNA purification kit (Invitrogen, Carlsbad, CA) was used to generate complementary DNA (cDNA) using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA) following the manufacturers' suggested protocols. The resulting cDNA was used as template in a PCR with SVNaV-specific primers, SVNaV-f1 and SVNaV-r1 (3). PCRs of two of the six Wisconsin samples and two Iowa samples were positive. Amplification products were not detected in the other five samples. The amplification products from the four strongly positive samples were purified using the Wizard SV Gel and PCR Purification Kit (Promega, Madison, WI) following the manufacturer's suggested protocol and were subjected to automated sequencing (University of Wisconsin Biotechnology Center or Iowa State University, DNA Sequencing Facilities). BLASTn (1) alignments of the 915-bp consensus sequence revealed 98% and >99% identity of the Wisconsin and Iowa samples, respectively, with the 'S' segment of the SVNaV 'TN' isolate (GenBank Accession No. GU722319.1). Samples from the same leaf tissue used above, were subjected to serological tests for SVNaV using antigen coated-indirect ELISA (3). Asymptomatic soybeans grown in the greenhouse were used as a source of leaves for negative controls. These tests confirmed the presence of SVNaV in eight symptomatic soybean leaflets collected in Wisconsin and Iowa. The asymptomatic control and one Iowa sample, which was also PCR-negative, were also negative by serological testing. Six additional samples from soybean fields in as many Wisconsin counties (Fond Du Lac, Grant, Green, Juneau, Richland, Rock) tested positive for SVNaV using specific primers that amplify the 'L' segment (4). The sequenced amplification products (297-bp) showed 99 to 100% homology to the L segment of the TN isolate (GU722317.1). To our knowledge, this is the first report of SVNaV associated with soybean and the first report of SVND in Wisconsin and Iowa. Considering that little is known about SVNaV, it is assumed that it is like other Tospoviruses and can cause significant yield loss (4). Soybean is a major cash crop for Wisconsin and Iowa, and infection by SVNaV could result in potential yield loss in years where epidemics begin early and at a high initial inoculum level. References: (1) S. F. Altschul et al. J. Mol. Biol. 215:403, 1990. (2) G. L. Hartman et al. Compendium of Soybean Diseases, 4th ed, 1999. (3) B. Khatabi et al. Eur. J. Plant Pathol. 133:783, 2012. (4) J. Zhou et al. Virus Genes 43:289, 2011.

Detection and variability of aster yellows phytoplasma titer in its insect vector, Macrosteles quadrilineatus (Hemiptera: Cicadellidae).

The aster yellows phytoplasma (AYp) is transmitted by the aster leafhopper, Macrosteles quadrilineatus Forbes, in a persistent and propagative manner. To study AYp replication and examine the variability of AYp titer in individual aster leafhoppers, we developed a quantitative real-time polymerase chain reaction assay to measure AYp concentration in insect DNA extracts. Absolute quantification of AYp DNA was achieved by comparing the amplification of unknown amounts of an AYp target gene sequence, elongation factor TU (tuf), from whole insect DNA extractions, to the amplification of a dilution series containing known quantities of the tuf gene sequence cloned into a plasmid. The capabilities and limitations of this method were assessed by conducting time course experiments that varied the incubation time of AYp in the aster leafhopper from 0 to 9 d after a 48 h acquisition access period on an AYp-infected plant. Average AYp titer was measured in 107 aster leafhoppers and, expressed as Log10 (copies/insect), ranged from 3.53 (+/- 0.07) to 6.26 (+/- 0.11) occurring at one and 7 d after the acquisition access period. AYp titers per insect and relative to an aster leafhopper chromosomal reference gene, cp6 wingless (cp6), increased approximately 100-fold in insects that acquired the AYp. High quantification cycle values obtained for aster leafhoppers not exposed to an AYp-infected plant were interpreted as background and used to define a limit of detection for the quantitative real-time polymerase chain reaction assay. This method will improve our ability to study biological factors governing AYp replication in the aster leafhopper and determine if AYp titer is associated with frequency of transmission.

A soluble form of the Tomato spotted wilt virus (TSWV) glycoprotein G(N) (G(N)-S) inhibits transmission of TSWV by Frankliniella occidentalis.

Tomato spotted wilt virus (TSWV) is an economically important virus that is transmitted in a persistent propagative manner by its thrips vector, Frankliniella occidentalis. Previously, we found that a soluble form of the envelope glycoprotein G(N) (G(N)-S) specifically bound thrips midguts and reduced the amount of detectable virus inside midgut tissues. The aim of this research was to (i) determine if G(N)-S alters TSWV transmission by thrips and, if so, (ii) determine the duration of this effect. In one study, insects were given an acquisition access period (AAP) with G(N)-S mixed with purified virus and individual insects were assayed for transmission. We found that G(N)-S reduced the percent of transmitting adults by eightfold. In a second study, thrips were given an AAP on G(N)-S protein and then placed on TSWV-infected plant material. Individual insects were assayed for transmission over three time intervals of 2 to 3, 4 to 5, and 6 to 7 days post-adult eclosion. We observed a significant reduction in virus transmission that persisted to the same degree throughout the time course. Real-time reverse transcription polymerase chain reaction analysis of virus titer in individual insects revealed that the proportion of thrips infected with virus was reduced threefold when insects were preexposed to the G(N)-S protein as compared to no exposure to protein, and nontransmitters were not infected with virus. These results demonstrate that thrips transmission of a tospovirus can be reduced by exogenous viral glycoprotein.

Precise efficiency calibration of an HPGe detector: source measurements and Monte Carlo calculations with sub-percent precision.

With the goal of measuring precise gamma-ray intensities for short-lived (< 5 s) accelerator-produced activities, we have calibrated the efficiency of an HPGe detector between 53 and 1836keV to sub-percent precision with a combination of source measurements and Monte Carlo calculations. Using known or independently measured detector dimensions, we have achieved both relative and absolute agreement (the latter, to 0.1%) between the calculated and measured efficiencies with only two adjustable detector parameters, the thicknesses of the contact dead layers.

Use of an intergenic region in Pseudomonas syringae pv. syringae B728a for site-directed genomic marking of bacterial strains for field experiments.

To construct differentially-marked derivatives of our model wild-type strain, Pseudomonas syringae pv. syringae B728a (a causal agent of bacterial brown spot disease in snap bean plants), for field experiments, we selected a site in the gacS-cysM intergenic region for site-directed insertion of antibiotic resistance marker cassettes. In each of three field experiments, population sizes of the site-directed chromosomally marked B728a derivatives in association with snap bean plants were not significantly different from that of the wild-type strain. Inserts of up to 7 kb of DNA in the intergenic region did not measurably affect fitness of B728a in the field. The site is useful for site-directed genomic insertions of single copies of genes of interest.

Genetic evidence that loss of virulence associated with gacS or gacA mutations in Pseudomonas syringae B728a does not result from effects on alginate production.

Mutations in the global regulatory genes gacS and gacA render Pseudomonas syringae pv. syringae strain B728a completely nonpathogenic in foliar infiltration assays on bean plants. It had been previously demonstrated that gac genes regulate alginate production in Pseudomonas species, while other published work indicated that alginate is involved in the pathogenic interaction of P. syringae on bean plants. Together, these results suggested that the effects of gacS and gacA mutations on virulence in B728a might stem directly from a role in regulating alginate. In this report, we confirm a role for gac genes in both algD expression and alginate production in B728a. However, B728a mutants completely devoid of detectable alginate were as virulent as the wild-type strain in our assay. Thus, factors other than, or in addition to, a deficiency of alginate must be involved in the lack of pathogenicity observed with gacS and gacA mutants.

Transposon insertion in the ftsK gene impairs in planta growth and lesion-forming abilities in Pseudomonas syringae pv. syringae B728a.

A Tn5 insertion in the ftsK gene of Pseudomonas syringae pv. syringae B728a impaired brown spot lesion formation on Phaseolus vulgaris, the ability to grow within bean leaves, and swarming ability on semisolid agar. Plasmids containing the ftsK gene were sufficient to complement the original Tn5 mutant for lesion formation and swarming and partially restored in planta growth.

Role of the Hrp type III protein secretion system in growth of Pseudomonas syringae pv. syringae B728a on host plants in the field.

hrp genes are reportedly required for pathogenicity in Pseudomonas syringae pv. syringae (Pss) and other phytopathogenic bacterial species. A subset of these genes encodes a type III secretion system through which virulence factors are thought to be delivered to plant cells. In this study, we sought to better understand the role that hrp genes play in interactions of Pss with its host as they occur naturally under field conditions. Population sizes of hrp mutants with defects in genes that encode components of the Hrp secretion system (DeltahrcC::nptII and hrpJ:: OmegaSpc) and a protein secreted via the system (DeltahrpZ::nptII) were similar to B728a on germinating seeds. However, phyllosphere (i.e., leaf) population sizes of the hrcC and hrpJ secretion mutants, but not the hrpZ mutant, were significantly reduced relative to B728a. Thus, the Hrp type III secretion system, but not HrpZ, plays an important role in enabling Pss to flourish in the phyllosphere, but not the spermosphere. The hrcC and hrpJ mutants caused brown spot lesions on primary leaves at a low frequency when they were inoculated onto seeds at the time of planting. Pathogenic reactions also were found when the hrp secretion mutants were co-infiltrated into bean leaves with a non-lesion-forming gacS mutant of B728a. In both cases, the occurrence of disease was associated with elevated population sizes of the hrp secretion mutants. The role of the Hrp type III secretion system in pathogenicity appears to be largely mediated by its requirement for growth of Pss in the phyllosphere. Without growth, disease does not occur.

Swarming by Pseudomonas syringae B728a requires gacS (lemA) and gacA but not the acyl-homoserine lactone biosynthetic gene ahlI.

Pseudomonas syringae pv. syringae B728a, a causal agent of bacterial brown spot on snap beans, swarms with a characteristic dendritic pattern on semisolid (0.4%) agar plates. Filamentation of swarming cells of B728a was not observed. Mutations in either the gacS (formerly lemA) or gacA gene of B728a eliminate the ability of this P. syringae isolate to swarm without obvious effects on bacterial motility. Three field isolates showed a similar dependence on gacS for swarming. Since gacS and gacA mutants are known to be deficient in N-acyl-L-homoserine lactone (acyl-HSL) production, a mutant was constructed by disruption of the ahlI gene of B728a. This mutant did not make any acyl-HSL detectable by the Agrobacterium traG::lacZ reporter system, yet was unaffected in its ability to swarm. Other phenotypes of gacS and gacA mutations were similarly unaffected in the ahlI mutant.

A newly identified regulator is required for virulence and toxin production in Pseudomonas syringae.

The genes lemA (which we here redesignate gacS) and gacA encode members of a widely conserved two-component regulatory system. In Pseudomonas syringae strain B728a, gacS and gacA are required for lesion formation on bean, as well as for the production of protease and the toxin syringomycin. A gene, designated salA, was discovered that restored syringomycin production to a gacS mutant when present on a multiple-copy plasmid. Disruption of chromosomal salA resulted in loss of syringomycin production and lesion formation in laboratory assays. Sequence analysis of salA suggests that it encodes a protein with a DNA-binding motif but without other significant similarity to proteins in current databases. Chromosomal reporter fusions revealed that gacS and gacA positively regulate salA, that salA upregulates its own expression and that salA positively regulates the expression of a syringomycin biosynthetic gene, syrB. Loss of syringomycin production does not account for the salA mutant's attenuated pathogenicity, as a syrB mutant was found to retain full virulence. The salA gene did not similarly suppress the protease deficient phenotype of gacS mutants, nor were salA mutants affected for protease production. A gacS/gacA-dependent homoserine lactone activity as detected by bioassay was also unaffected by the disruption of salA. Thus, salA appears to encode a novel regulator that activates the expression of at least two separate genetic subsets of the gacS/gacA regulon, one pathway leading to syringomycin production and the other resulting in plant disease.

Multiple loci of Pseudomonas syringae pv. syringae are involved in pathogenicity on bean: restoration of one lesion-deficient mutant requires two tRNA genes.

A mutational analysis of lesion-forming ability was undertaken in Pseudomonas syringae pv. syringae B728a, causal agent of bacterial brown spot disease of bean. Following a screen of 6,401 Tn5-containing derivatives of B728a on bean pods, 26 strains that did not form disease lesions were identified. Nine of the mutant strains were defective in the ability to elicit the hypersensitive reaction (HR) and were shown to contain Tn5 insertions within the P. syringae pv. syringae hrp region. Ten HR+ mutants were defective in the production of the toxin syringomycin, and a region of the chromosome implicated in the biosynthesis of syringomycin was deleted in a subset of these mutants. The remaining seven lesion-defective mutants retained the ability to produce protease and syringomycin. Marker exchange mutagenesis confirmed that the Tn5 insertion was causal to the mutant phenotype in several lesion-defective, HR+ strains. KW239, a lesion- and syringomycin-deficient mutant, was characterized at the molecular level. Sequence analysis of the chromosomal region flanking the Tn5 within KW239 revealed strong similarities to a number of known Escherichia coli gene products and DNA sequences: the nusA operon, including the complete initiator tRNA(Met) gene, metY; a tRNA(Leu) gene; the tpiA gene product; and the MrsA protein. Removal of sequences containing the two potential tRNA genes prevented restoration of mutant KW239 in trans. The Tn5 insertions within the lesion-deficient strains examined, including KW239, were not closely linked to each other or to the lemA or gacA genes previously identified as involved in lesion formation by P. syringae pv. syringae.

Contribution of the Regulatory Gene lemA to Field Fitness of Pseudomonas syringae pv. syringae.

In Pseudomonas syringae pv. syringae, lemA is required for brown spot lesion formation on snap bean and for production of syringomycin and extracellular proteases (E. M. Hrabak and D. K. Willis, J. Bacteriol. 174: 3011-3022, 1992; E. M. Hrabak and D. K. Willis, Mol. Plant-Microbe Interact. 6:368-375, 1993; D. K. Willis, E. M. Hrabak, J. J. Rich, T. M. Barta, S. E. Lindow, and N. J. Panopoulos, Mol. Plant-Microbe Interact. 3:149-156, 1990). The lemA mutant NPS3136 (lemA1::Tn5) was previously found to be indistinguishable from its pathogenic parent B728a in its ability to grow when infiltrated into bean leaves of plants maintained under controlled environmental conditions (Willis et al., Mol. Plant-Microbe Interact. 3:149-156, 1990). We compared population sizes of NPS3136 and B728aN (a Nal(supr) clone of wild-type B728a) in two field experiments to determine the effect of inactivation of lemA on the fitness of P. syringae pv. syringae. In one experiment, the bacterial strains were spray inoculated onto the foliage of 25-day-old bean plants. In the other, seeds were inoculated at the time of planting. In both experiments, the strains were inoculated individually and coinoculated in a 1:1 ratio. NPS3136 and B728aN achieved similar large population sizes on germinating seeds. However, in association with leaves, population sizes of NPS3136 were diminished relative to those of B728aN in both experiments. Thus, lemA contributed significantly to the fitness of P. syringae pv. syringae in association with bean leaves but not on germinating seeds under field conditions. When NPS3136 was coinoculated with B728aN, the mutant behaved as it did when inoculated alone. However, population sizes of B728aN in the coinoculation treatment were much lower than those when it was inoculated alone. Inactivation of the lemA gene appeared to have rendered the mutant suppressive to B728aN.

Suppression of a sensor kinase-dependent phenotype in Pseudomonas syringae by ribosomal proteins L35 and L20.

The lemA gene of Pseudomonas syringae pv. syringae encodes the sensor kinase of a bacterial two-component signal transduction system. Phenotypes that are lemA dependent in P. syringae include lesion formation on bean and production of extracellular protease and the antibiotic syringomycin. Recently, the gacA gene has been identified as encoding the response regulator of the lemA regulon. To identify additional components that interact with LemA, suppressors of a lemA mutation were sought. A locus was identified that, when present in multiple copies, restores extracellular protease production to a lemA insertion mutant of P. syringae pv. syringae. This locus was found to encode the P. syringae homologs of translation initiation factor IF3 and ribosomal proteins L20 and L35 of Escherichia coli and other bacteria. Deletion analysis and data from Western immunoblots with anti-IF3 antiserum suggest that protease restoration does not require IF3. Deletion of both the L35 and L20 genes resulted in loss of protease restoration, whereas disruption of either gene alone increased protease restoration. Our results suggest that overexpression of either L20 or L35 is sufficient for protease restoration. It is unclear how alteration of ribosomal protein expression compensates in this instance for loss of a transcriptional activator, but a regulatory role for L20 and L35 apart from their function in the ribosome may be indicated.

Genetic evidence that the gacA gene encodes the cognate response regulator for the lemA sensor in Pseudomonas syringae.

Mutational analysis of the bean-pathogenic Pseudomonas syringae pv. syringae strain B728a has led to the genetic identification of the gacA gene as encoding the response regulator for the unlinked lemA sensor kinase. The analysis of a collection of spontaneous mutants of P. syringae pv. syringae suggested that the gacA gene was involved in lesion formation and the production of protease and syringomycin. The gacA gene originally was identified as a regulator of extracellular antibiotic production by Pseudomonas fluorescens, and the predicted GacA protein is a member of the FixJ family of bacterial response regulators. The sequence of the putative B728a GacA protein revealed 92% identity with the P. fluorescens GacA protein. An insertional mutation within the P. syringae pv. syringae gacA gene abrogated lesion formation on beans, production of extracellular protease, and production of the toxin syringomycin, the same phenotypes affected by a lemA mutation. DNA sequence analysis identified the P. syringae pv. syringae uvrC gene immediately downstream of the gacA gene, an arrangement conserved in P. fluorescens and Escherichia coli. The gacA insertional mutant was sensitive to UV, presumably because of polarity on transcription of the downstream uvrC gene. Southwestern (DNA-protein) analysis revealed that the lemA and gacA genes were required for the full expression of a DNA binding activity.

DNA sequence and transcriptional analysis of the tblA gene required for tabtoxin biosynthesis by Pseudomonas syringae.

The tblA gene of Pseudomonas syringae is required for tabtoxin biosynthesis and is under the control of a regulatory gene, lemA. We have determined the nucleotide sequence of the tblA gene and identified the 5' end of the tblA gene transcript. The sequence of the tblA gene was identified to that of the recently reported open reading frame 1 gene of the tabA region of the BR2 chromosome. The open reading frame of the tblA gene potentially encodes a protein of 231 amino acids. mRNA from the tblA gene was detected at all phases of cells grown in minimal medium. This result is correlated with the constitutive production of tabtoxinine-beta-lactam (the biologically active part of the toxin) by P. syringae BR2R in minimal medium, as quantitated by a phenylisothiocyanate derivatization method.

The lemA gene required for pathogenicity of Pseudomonas syringae pv. syringae on bean is a member of a family of two-component regulators.

The lemA gene of the plant pathogen Pseudomonas syringae pv. syringae is required for disease lesion formation on bean plants. Cosmid clones that complemented a lemA mutant in trans were isolated previously. The lemA gene was localized by subcloning and transposon mutagenesis. The lemA region and flanking DNA were sequenced, and an open reading frame of 2.7 kb was identified. The nucleotide and predicted amino acid sequences of the lemA gene showed sequence similarity to a family of prokaryotic two-component regulatory proteins. Unlike most of the previously described two-component systems, the lemA gene product contained homology to both components in one protein. Mutations introduced upstream and downstream of the lemA gene failed to locate a gene for a second protein component but identified the putative cysM gene of P. syringae pv. syringae. The cysM gene was located upstream of the lemA gene and was divergently transcribed. The lemA gene product was expressed at low levels in P. syringae pv. syringae and appeared to be positively auto-regulated.

Regulation of tabtoxin production by the lemA gene in Pseudomonas syringae.

Pseudomonas syringae pv. coronafaciens, a pathogen of oats, was mutagenized with Tn5 to generate mutants defective in tabtoxin production. From a screen of 3,400 kanamycin-resistant transconjugants, seven independent mutants that do not produce tabtoxin (Tox-) were isolated. Although the Tn5 insertions within these seven mutants were linked, they were not located in the previously described tabtoxin biosynthetic region of P. syringae. Instead, all of the insertions were within the P. syringae pv. coronafaciens lemA gene. The lemA gene is required by strains of P. syringae pv. syringae for pathogenicity on bean plants (Phaseolus vulgaris). In contrast to the phenotype of a P. syringae pv. syringae lemA mutant, the Tox- mutants of P. syringae pv. coronafaciens were still able to produce necrotic lesions on oat plants (Avena sativa), although without the chlorosis associated with tabtoxin production. Northern (RNA) hybridization experiments indicated that a functional lemA gene was required for the detection of a transcript produced from the tblA locus located in the tabtoxin biosynthetic region. Marker exchange mutagenesis of the tblA locus resulted in loss of tabtoxin production. Therefore, both the tblA and lemA genes are required for tabtoxin biosynthesis, and the regulation of tabtoxin production by lemA probably occurs at the transcriptional level.

Pathovar-specific requirement for the Pseudomonas syringae lemA gene in disease lesion formation.

The lemA gene is conserved among strains and pathovars of Pseudomonas syringae. In P. syringae pv. syringae B728a, a causal agent of bacterial brown spot disese of bean, the lemA gene is required for lesion formation on leaves and pods. Using lemA-containing DNA as a probe, we determined that 80 P. syringae pv. syringae strains isolated from bean leaves could be grouped into seven classes based on restriction fragment length polymorphism. Marker exchange mutagenesis showed that the lemA gene was required for lesion formation by representative strains from each restriction fragment length polymorphism class. Hybridization to the lemA locus was detected within six different P. syringae pathovars and within Pseudomonas aeruginosa. Interestingly, a lemA homolog was present and functional within the nonpathogenic strain P. syringae Cit7. We cloned a lemA homolog from a genomic library of P. syringae pv. phaseolicola NPS3121, a causal agent of halo blight of bean, that restored lesion formation to a P. syringae pv. syringae lemA mutant. However, a lemA mutant P. syringae pv. phaseolicola strain retained the ability to produce halo blight disease symptoms on bean plants. Therefore, the lemA gene played an essential role in disease lesion formation by P. syringae pv. syringae isolates, but was not required for pathogenicity of a P. syringae pv. phaseolicola strain.

Cloning and expression of the tabtoxin biosynthetic region from Pseudomonas syringae.

Pseudomonas syringae BR2, a causal agent of bean wildfire, was subjected to Tn5 mutagenesis in an effort to isolate mutants unable to produce the beta-lactam antibiotic tabtoxin. Three of the tabtoxin-minus (Tox-) mutants generated appeared to have physically linked Tn5 insertions and retained their resistance to the active toxin form, tabtoxnine-beta-lactam (T beta L). The wild-type DNA corresponding to the mutated region was cloned and found to restore the Tn5 mutants to toxin production. The use of cloned DNA from the region as hybridization probes revealed that the region is highly conserved among tabtoxin-producing pathovars of P. syringae and that the region deletes at a relatively high frequency (10(-3)/CFU) in BR2. The Tox- deletion mutants also lost resistance to tabtoxinine-beta-lactam. A cosmid designated pRTBL823 restored toxin production and resistance to BR2 deletion mutants. This cosmid also converted the tabtoxin-naive P. syringae epiphyte Cit7 to toxin production and resistance, indicating that pRTBL823 contains a complete set of biosynthetic and resistance genes. Tox- derivatives of BR2 did not produce disease symptoms on bean. Clones that restored toxin production to both insertion and deletion mutants also restored the ability to cause disease. However, tabtoxin-producing Cit7 derivatives remained nonpathogenic on bean and tobacco, suggesting that tabtoxin production alone is not sufficient to cause disease.