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The association between baseline serum chloride levels and mortality in patients with severe AKI is unknown. Our aim was to investigate the relationship between baseline blood chloride levels and 28-day mortality in patients with AKI admitted to the ICU and to detect possible effect modifiers in this population.AKI patients with severe critical illness were extracted from the MIMICIV. During ICU admission, chloride levels were measured for the first time. Our primary outcome was 28-day mortality in patients with AKI after 24 hours in the ICU. Multivariable logistic regression was used to examine the association between three groups of chloride levels and 28-day mortality, and logistic regression with restricted cubic spline was applied to detect the non-linear trendy. RESULTSː A total of 24,166 patients with critically ill AKI were included in this retrospective cohort study. The total 28-day mortality rate in the ICU was 15.9%. Overall, there was a U-shaped relationship between baseline serum chloride levels and 28-day mortality (non-linear p0.001). Accordingly, patients with low serum chloride (96mEq/L) had a significantly increased risk of death compared to patients with normal serum chloride (96-108meq/L) (adjusted OR=1.96, 95% CI=1.70-2.27, P0.001). None of the variables, including age, gender, 24-hour fluid intake, continuous renal replacement therapy, ventilation, Atrial fibrillation, Sequential Organ Failure Assessment score and whether to measure lactate, significantly modified the association between lower chloride levels and 28-day mortality. CONCLUSIONSː Low serum chloride levels at baseline were associated with death at 28 days in intensive illnesses with AKI.

Genome sequences of a novel begomovirus infecting tomato on Guam were obtained using primer-walking and sequencing. The complete genome sequences are 2,750 nucleotides long with a typical monopartite organization and display less than 91% nucleotide sequence identity to other begomoviruses. A provisional name, tomato leaf curl Guam virus (ToLCGuV), is proposed.

Tomato planta macho viroid (TPMVd), including isolates previously designated as Mexican papita viroid (MPVd), causes serious disease on tomatoes in North America. Two predominant variants, sharing 93.8% sequence identity, incited distinct severe (MPVd-S) or mild (MPVd-M) symptoms on tomato. To identify virulence determinant factor, a series of chimeric infectious clones were generated using synthetic DNA approach to progressively replace each structural domain between the two variants. In bioassays on tomato 'Rutgers', three chimeras containing Terminal Left and Pathogenicity (MPVd-H1), Central (MPVd-H2), or Variable (MPVd-H3) of MPVd-S, incited mild to intermediate symptoms. However, a chimera containing Terminal Right (TR) of MPVd-S (MPVd-H4) incited severe symptoms. Only one base-pair mutation in the TR domain between MPVd-M (176U:A185) and MPVd-S (174G:C183) was identified. A reciprocal mutant (MPVd-H5) rendered the chimeric viroid mild on tomato. This single base-pair in the TR domain was determined as the virulence determinant factor for TPMVd.

In early 2013, greenhouse cucumber growers in Alberta, Canada, observed virus-like disease symptoms on mini-cucumber (Cucumis sativus) crops (e.g., ‘Picowell’). Two types of symptoms were commonly observed, green mottle mosaic and necrotic spots. In the early infection, young leaves of infected cucumber plants displayed light green mottle and blisters. The infected plants were stunted in growth, with darker green blisters and green mottle mosaic symptoms on mature leaves. Disease incidence varied from one greenhouse to another. In some severe cases, diseased plants were widely distributed inside the greenhouse, resulting in 10 to 15% yield losses based on grower's estimation. Nine symptomatic samples were collected and subjected to total RNA isolation using the TRIzol reagent (Invitrogen, Carlsbad, CA). Laboratory analyses were conducted using real-time RT-PCR systems for Cucumber green mottle mosaic virus (CGMMV) (1), Melon necrotic spot virus (MNSV, Ling, unpublished), and Squash mosaic virus (SqMV) (3). All nine samples were positive for CGMMV and seven of them were in mixed infections with MNSV. Two samples were selected for validation for the presence of CGMMV using conventional RT-PCR (2) with a new primer set (CGMMVMP F1: 5′-ATGTCTCTAAGTAAGGTGTC-3′ and CGMMV3′UTR R1: 5′-TGGGCCCCTACCCGGGG-3′) and two previous online published primer sets, one for CGMMV MP (5′ TAAGTTTGCTAGGTGTGATC-3′, GenBank Accession No. AJ250104 and 5′ ACATAGATGTCTCTAAGTAAG-3′, AJ250105), and another for CGMMV CP (5′ ACCCTCGAAACTAAGCTTTC-3′, AJ243351 and 5′ GAAGAGTCCAGTTCTGTTTC-3′, AJ243352). The expected sizes of RT-PCR products were obtained and sequenced directly. Sequences from these three products overlapped and generated a 1,282-bp contig (KF683202). BLASTn analysis to the NCBI database showed 99% sequence identity to CGMMV isolates identified in Asia, including China (GQ277655, KC852074), India (DQ767631), Korea (AF417243), Myanmar (AB510355), and Taiwan (HQ692886), but only 92% sequence identity to other CGMMV isolates identified in Europe, including Spain (GQ411361) and Russia (GQ495274), and 95% to CGMMV isolate from Israel (KF155231). The strong sequence identity to the CGMMV Asian isolates suggests that the Canadian CGMMV isolate identified in Alberta was likely of Asian origin. In two bioassay experiments using one sample prepared in 0.01 M phosphate buffer, the similar green mottle mosaic symptoms were observed on systemic leaves in the mechanically inoculated plants and the presence of CGMMV, but not MNSV, was confirmed through real-time RT-PCR on four different cucurbits, including three Cucumis sativus cultivars (six plants in ‘Marketer,’ five plants in ‘Poinsett 76,’ six plants in ‘Straight 8’), seven plants of C. melo ‘Athena,’ six plants of C. metulifer (PI201681), and two plants of Citrullus lanatus ‘Charleston Gray.’ To our knowledge, CGMMV has only been reported in Asia, Europe, and the Middle East, and this is the first report of CGMMV in the American continents. CGMMV is highly contagious and is seed borne on cucurbits. With the increasing trend in growing grafted watermelon and other cucurbits in the United States and elsewhere, it is even more important now that a vigilant seed health test program for CGMMV should be implemented. References: (1) H. Chen et al. J. Virol. Methods 149:326, 2008. (2) K.-S. Ling et al. Plant Dis. 92:1683, 2008. (3) K.-S. Ling et al. J. Phytopathol. 159:649, 2011.

Squash mosaic virus (SqMV), a seedborne virus, belongs to the genus Comovirus in the subfamily Comovirinae of the family Secoviridae. SqMV has a bipartite single-stranded RNA genome (RNA1 and RNA2) encapsidated separately with two capsid proteins. With the recent identification of a third genotype in SqMV, a greater genetic diversity with only 88 to 89% sequence identity among them are recognized. With the existence of genetic diversity, a previously developed quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) failed to detect isolates in this new genotype. Therefore, it was necessary to create a new qRT-PCR that would react with all SqMV isolates in three different genotypes. From a multiple sequence alignment of the available SqMV sequences in GenBank, a conserved sequence segment in the 3′ untranslated region of RNA2 was identified for primer and probe design. A new qRT-PCR was developed, which provided broad-spectrum reactions to SqMV isolates, including those from the newly recognized third genotype. To improve the reliability in determining the sample quality and result interpretation, an internal amplification control with an endogenous gene sequence (18S ribosomal RNA) was successfully incorporated to develop a duplex qRT-PCR system that was useful for seed health test.

Tomato mottle mosaic virus (ToMMV) was first identified in 2013 as a novel tobamovirus infecting tomatoes in Mexico. In just a few years, ToMMV has been identified in several countries around the world, including the United States. In the present study, we characterized the molecular, serological, and biological properties of ToMMV and developed a species-specific RT-PCR to detect three tomato-infecting tobamoviruses: Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), and ToMMV. Previously, ToMMV has been reported in Florida and New York. In this study, we made two new reports on the occurrences of ToMMV on tomatoes in California and South Carolina. Their complete genome sequences were obtained and their genetic relationships to other tobamoviruses evaluated. In host range studies, some differential responses in host plants were also identified between ToMMV and ToMV. To alleviate cross-serological reactivity among the tomato-infecting tobamoviruses, a new multiplex RT-PCR was developed to allow for species-specific detection and identification of TMV, ToMV, and ToMMV. In addition, we observed resistance breaking by ToMMV on selected tomato cultivars that were resistant to ToMV. This has caused serious concerns to tomato growers worldwide. In conclusion, the characterization in molecular and biological properties of ToMMV would provide us with fundamental knowledge to manage this emerging virus on tomato and other solanaceous crops in the U.S. and around the world.

Tomato necrotic stunt virus (ToNStV) is an emerging potyvirus that causes severe stunting to infected tomato plants. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for sensitive detection of ToNStV. The sensitivity of RT-LAMP was comparable to that of conventional RT-PCR, with detection of ToNStV in a reaction containing only 8 pg of total tomato RNA or with 1:20,000 dilution of crude tissue extract. This assay was able to detect ToNStV in a broad range of solanaceous plant species. The RT-LAMP for ToNStV was specific with no cross-reactivity to other potyviruses (i.e. Potato virus Y and Tobacco etch virus), as well as several other common tomato viruses. RT-LAMP should complement RT-PCR and real-time RT-PCR assays reported previously, with a potential to provide a simple, rapid, and sensitive field diagnostic method for ToNStV.

The newly identified tomato necrotic stunt virus (ToNStV), with a genome of 10,057 nucleotides, had 60 % nucleotide sequence and 70 % coat protein amino acid sequence identity to other potyviruses. Analysis of a second ToNStV isolate revealed two single nucleotide substitutions. ToNStV induced stunting and chlorotic to necrotic leaf symptoms on tomato plants. In host-range assays, ToNStV systemically infected many species in the family Solanaceae and locally infected some species in the families Amaranthaceae, Brassicaceae and Cucurbitaceae. A sensitive and reliable real-time RT-PCR assay was developed using simply prepared crude tissue extract.

Accurate detection of viruses in plants and animals is critical for agriculture production and human health. Deep sequencing and assembly of virus-derived small interfering RNAs has proven to be a highly efficient approach for virus discovery. Here we present VirusDetect, a bioinformatics pipeline that can efficiently analyze large-scale small RNA (sRNA) datasets for both known and novel virus identification. VirusDetect performs both reference-guided assemblies through aligning sRNA sequences to a curated virus reference database and de novo assemblies of sRNA sequences with automated parameter optimization and the option of host sRNA subtraction. The assembled contigs are compared to a curated and classified reference virus database for known and novel virus identification, and evaluated for their sRNA size profiles to identify novel viruses. Extensive evaluations using plant and insect sRNA datasets suggest that VirusDetect is highly sensitive and efficient in identifying known and novel viruses. VirusDetect is freely available at http://bioinfo.bti.cornell.edu/tool/VirusDetect/.

The complete genome sequence of a Southern tomato virus (STV) isolate on tomato plants in a seed production field in Bangladesh was obtained for the first time using next-generation sequencing. The identified isolate, STV_BD-13, shares a high degree of sequence identity (99%) with several known STV isolates worldwide.

The complete genome sequence of an isolate of tomato mottle mosaic virus (ToMMV) infecting tomatoes in New York was obtained using small RNA (sRNA) deep sequencing. ToMMV_NY-13 shared 99% sequence identity with isolates from Mexico and Florida. Broader distribution of this emerging virus is a cause for concern to the tomato industry.

Particle bombardment has been used for soybean transformation for more than 20 yr, but the integration and segregation of transgene inserts in the soybean genome have not been clearly documented. Over the past 5 yr, we processed several hundred transgenic events. In each experiment, the expression cassettes of the gene of interest (GOI) and hygromycin selectable marker gene (SMG) were co-bombarded into soybean at a 1:1 molecular ratio. More than 75% of hygromycin-resistant events also carried the GOI. Molecular analysis of transgenic plants revealed that most events carried multiple inserts of the GOI and the SMG. The GOI and the SMG were linked in selfed T1 and T2 progeny. Segregation analysis of progeny indicated that, in over 98% of the transgenic events, the multiple inserts of the GOI were integrated into the same genetic locus resulting in a 3:1 segregation ratio. Furthermore, the multiple inserts of the GOI are transmitted into succeeding generations, and no recombinants were found. These data indicate that in soybean plants, co-bombarded genes are preferentially integrated and stably segregated as a single genetic locus.

Most plants encode a limited set of polygalacturonase inhibitor (PGIP) genes that may be involved in aspects of plant development, but more importantly in the inactivation of polygalacturonases (PG) secreted by pathogens. Previously, we characterized two Brassica napus PGIP genes, BnPgip1 and BnPgip2, which were differentially expressed in response to pathogen infection and wounding. Here we report that the B. napus genome encodes a set of at least 16 PGIP genes that are similar to BnPgip1 or BnPgip2. This is the largest Pgip gene family reported to date. Comparison of the BnPGIPs revealed several sites within the xxLxLxx region of leucine rich repeats that form beta-sheets along the interacting face of the PGIP that are hypervariable and represent good candidates for generating PGIP diversity. Characterization of the regulatory regions and RT-PCR studies with gene-specific primers revealed that individual genes were differentially responsive to pathogen infection, mechanical wounding and signaling molecules. Many of the BnPgip genes responded to infection by the necrotic pathogen, Sclerotinia sclerotiorum; however, these genes were also induced either by jasmonic acid, wounding and salicylic acid or some combination thereof. The large number of PGIPs and the differential manner in which they are regulated likely ensures that B. napus can respond to attack from a broad spectrum of pathogens and pests.

The complete genome sequence of Southern tomato virus (STV), a double-stranded RNA virus that affects tomato in China, was determined using small RNA deep sequencing. This Chinese isolate shares 99% sequence identity to other isolates from Mexico, France, Spain, and the United States. This is the first report of STV infecting tomatoes in Asia.

The complete genome sequence (6,423 nucleotides [nt]) of an emerging cucumber green mottle mosaic virus (CGMMV) isolate on cucumber in North America was determined through deep sequencing of small (sRNA) and rapid amplification of cDNA ends. The virus shares 99% nucleotide sequence identity with the Asian genotype but only 90% with the European genotype.

The complete genome sequence of a new isolate of squash mosaic virus (SqMV) infecting squash plants in Spain was obtained using deep sequencing of small RNAs. The low nucleotide sequence identities, with only 87 to 88% for RNA1 and 84 to 86% for RNA2 to known SqMV isolates, suggested that this isolate belongs to a novel genotype.

We report here the complete genome sequence of an emerging genotype of tobacco streak virus (TSV) infecting zucchini squash in Florida (TSV_FL13-07), obtained using deep sequencing of short RNAs (sRNAs) and validation by Sanger sequencing. TSV_FL13-07 shares only

Five major and several minor PG isoenzymes were identified in a Sclerotinia sclerotiorum isolate from Brassica napus by isoelectric focusing and pectin gel overlays. Using a combination of degenerate PCR and expressed sequence tags (ESTs) four endo-polygalacturonase (PG) genes, designated as sspg1d, sspg3, sspg5, and sspg6, and two exo-PG genes, ssxpg1 and ssxpg2, were identified. SSPG1d is a member of the PG gene family previously described by Fraissinet-Tachet et al. [Curr. Genet. 29 (1995) 96]. The mature SSPG1d is a neutral PG, whereas fully processed SSPG3, SSPG5, and SSPG6 are acidic enzymes. Under saprophytic growth conditions, sspg1d, sspg3, sspg5, and ssxpg1 expression was induced by pectin and galacturonic acid and subject to catabolite repression by glucose. Conditions could not be identified under which sspg6 or ssxpg2 were expressed well. Transfer of mycelia from liquid media to solid substrates induced expression of sspg1d suggesting that it may also be regulated by thigmotrophic interactions. Under pathogenic conditions, sspg1d was highly expressed during infection. sspg3 was also expressed during infection, albeit at lower levels than sspg1d, whereas sspg5, sspg6, and ssxpg1 were expressed only weakly.

Plants encode a distinct set of polygalacturonase inhibitory proteins (PGIPs) that function to inhibit polygalacturonase enzymes produced by soft-rot fungal pathogens. We characterized two PGIP-encoding genes ( Bnpgip1 and Bnpgip2) from Brassica napus DH12075 (a double-haploid line derived from a cross between 'Crésor' and 'Westar'). The two proteins exhibit 67.4% identity at the amino acid level and contain 10 imperfect leucine-rich repeats. The pgip genes are present as a small multigene family in B. napus with at least four members. Bnpgip1 and Bnpgip2 are constitutively expressed in roots, stems, flower buds and open flowers. In mature leaf tissue, different levels of induction were observed in response to biotic and abiotic stresses. Bnpgip1 expression was highly responsive to flea beetle feeding and mechanical wounding, weakly responsive to Sclerotinia sclerotiorum infection and exposure to cold but not to dehydration. Conversely, Bnpgip2 expression was strongly induced by S. sclerotiorum infection and to a lesser degree by wounding but not by flea beetle feeding. Application of jasmonic acid to leaves induced both Bnpgip1 and Bnpgip2 gene expression; however, salicylic acid did not activate either gene. Taken together, these results suggest that separate pathways regulate Bnpgip1 and Bnpgip2, and that their roles in plant development or resistance to biotic and abiotic stress differ.

Small interfering RNA (siRNA) duplexes are short (usually 21 to 24 bp) double-stranded RNAs (dsRNAs) with several overhanging nucleotides at both 5′- and 3′-ends. It has been found that siRNA duplexes bind the RNA-induced silencing complex (RISC) and cleave the sense strands with endonucleases. In this study, for the first time, we detected siRNA duplexes induced by plant viruses on a large scale using next-generation sequencing (NGS) data. In addition, we used the detected 21 nucleotide (nt) siRNA duplexes with 2 nt overhangs to construct a dataset for future data mining. The analytical results of the features in the detected siRNA duplexes were consistent with those from previous studies. The investigation of siRNA duplexes is useful for a better understanding of the RNA interference (RNAi) mechanism. It can also help to improve the virus detection based on the small RNA sequencing (sRNA-seq) technologies and to rationally design siRNAs for RNAi experiments.

Aspergillus flavus (Link:Fr.) infection and aflatoxin contamination of maize (Zea mays L.) grain are an extremely serious problem. Maize genotypes resistant to A. flavus attack are needed. Maize breeders and plant pathologists must identify resistance sources and incorporate resistance into adapted breeding material. Maize population GT-MAS:gk has been released for use as a resistance source. In this study, we surveyed the genetic variation in this population and made the breeders/plant pathologists aware of the heterogeneous nature in this maize population by using RAPD analysis and correlated the RAPD marker association with the resistance to A. flavus and aflatoxin production. Of 40 RAPD primers, only 15 gave sufficient numbers of reproducible and readily scored polymorphic bands suggesting that this population was highly homogeneous. However, genetic distances, ranging from 0.08 to 0.28 and averaging 0.17, suggest that there is variation within the population. Cluster analysis distinguished three major polymorphic groups. Laboratory bioassay revealed that group I contained the most resistant individuals, i.e., those with less aflatoxin production. Group II had the least resistance, and group III was intermediate. This study showed that the maize population GT-MAS:gk is heterogeneous and individuals are different in resistance to A. flavus and aflatoxin production. Resistance should be confirmed through progeny testing before further development. The RAPD marker OPX-04, which may be associated with the resistance trait, has been cloned and further characterization will be pursued.

Maysin, a C-glycosylflavone in maize silk, has insecticidal activity against corn earworm, Helicoverpa zea (Boddie), larvae. Sweet corn, Zea mays L., is a vulnerable crop to ear-feeding insects and requires pesticide protection from ear damage. This study was conducted to identify maize chromosome regions associated with silk maysin concentration and eventually to transfer and develop high silk maysin sweet corn lines with marker-assisted selection (MAS). Using an F2 population derived from SC102 (high maysin dent corn) and B31857 (low maysin sh2 sweet corn), we detected two major quantitative trait loci (QTL). It was estimated that 25.6% of the silk maysin variance was associated with segregation in the genomic region of npi286 (flanking to p1) on chromosome 1S. We also demonstrated that a1 on chromosome 3L had major contribution to silk maysin (accounted for 15.7% of the variance). Locus a1 has a recessive gene action for high maysin with the presence of functional p1 allele. Markers umc66a (near c2) and umc105a on chromosome 9S also were detected in this analysis with minor contribution. A multiple-locus model, which included npi286, a1, csu3 (Bin 1.05), umc245 (Bin 7.05), agrr21 (Bin 8.09), umc105a, and the epistatic interactions npi286 x a1, a1 x agrr21, csu3 x umc245, and umc105a x umc245, accounted for 76.3% of the total silk maysin variance. Tester crosses showed that at the a1 locus, SC102 has functional A1 alleles and B31857 has homozygous recessive a1 alleles. Individuals of (SC102 x B31857) x B31857 were examined with MAS and plants with p1 allele from SC102 and homozygous a1 alleles from B31857 had consistent high silk maysin. Marker-assisted selection seems to be a suitable method to transfer silk maysin to sweet corn lines to reduce pesticide application.

The complete genomic sequence of a new tobamovirus in tomatoes was determined through deep sequencing and assembly of small RNAs, then validated through Sanger sequencing. Based on the low sequence identity (≤85%) to known viruses and a close phylogenetic relationship to tobamoviruses, it was identified as a new species.

Background Pepino mosaic, once an emerging disease a decade ago, has become endemic on greenhouse tomatoes worldwide in recent years. Three distinct genotypes of Pepino mosaic virus (PepMV), including EU, US1 and CH2 have been recognized. Our earlier study conducted in 2006–2007 demonstrated a predominant EU genotype in Canada and United States. The objective of the present study was to monitor the dynamic of PepMV genetic composition and its current status in North America. Results Through yearly monitoring efforts in 2009–2012, we detected a dramatic shift in the prevalent genotype of PepMV from the genotype EU to CH2 in North America since early 2010, with another shift from CH2 to US1 occurring in Mexico only two years later. Through genetic diversity analysis using the coat protein gene, such genotype shifting of PepMV in North America was linked to the positive identification of similar sequence variants in two different commercial tomato seed sources used for scion and rootstock, respectively. To allow for a quick identification, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) system was developed and demonstrated to achieve a rapid identification for each of the three genotypes of PepMV, EU, US1 and CH2. Conclusion Through systemic yearly monitoring and genetic diversity analysis, we identified a linkage between the field epidemic isolates and those from commercial tomato seed lots as the likely sources of initial PepMV inoculum that resulted in genetic shifting as observed on greenhouse tomatoes in North America. Application of the genotype-specific RT-LAMP system would allow growers to efficiently determine the genetic diversity on their crops.

Fifty-two germplasm accessions of Chinese vegetable brassicas were analyzed using 112 random amplified polymorphic DNA (RAPD) markers. The array of material examined spanned a wide range of morphological, geographic, and genetic diversity, and included 30 accessions of Brassica rapa L. (Chinese cabbage, pakchoi, turnip, and broccoletto), 18 accessions of B. juncea (L.) Czern. (leaf, stem, and root mustards), and four accessions of B. oleracea L. ssp. alboglabra (Chinese kale). The RAPD markers unambiguously identified all 52 accessions. Nei-Li similarities were computed and used in unweighed pair group method using arithmetic means (UPGMA) cluster analyses. Accessions and subspecies were clustered into groups corresponding to the three species, but some accessions of some subspecies were most closely related to accessions belonging to other subspecies. Values for Nei-Li similarities suggest that Chinese cabbage is more likely to have been produced by hybridization of turnip and pakchoi than as a selection from either turnip or pakchoi alone. RAPD markers are a fast, efficient method for diversity assessment in Chinese vegetable brassicas that complements techniques currently in use in genetic resources collections. The crop brassicas comprise six economically important spe- cies with great genetic and morphological diversity. These plants yield edible roots, stems, leaves, buds, flowers, and seeds. In addition, some of the types are used as forage, sources of oil, or ornamental. The origin, evolution, taxonomy, and genomic relationships of

Because (i) origins and genealogies of vetiver grass have been poorly documented, and (ii) morphological uniformity and infrequent flowering precluded accurate identification of selected clones, an investigation was undertaken to link molecular methods with rigorous biometric analysis to discriminate accessions and clones. Successful DNA profiling is highly dependent on the reproducibility of the assay. Our ability to generate consistent results may have been a result of (i) genomic DNA quality, (ii) rigorous oligonucleotide primer selection, and (iii) DNA amplification protocol stringency. Because of the low level of replicate variability, we were able to bring a high level of resolution to the analysis of identity [...]

Sclerotinia sclerotiorum is a ubiquitous necrotrophic fungal pathogen capable of infecting a wide range of plants. To identify genes involved in fungal development and pathogenesis we generated 2232 expressed sequence tags (ESTs) from two cDNA libraries constructed using either mycelia grown in pectin medium or tissues from infected Brassica napus stems. A total of 774 individual fungal genes were identified of which 39 were represented only among the infected plant EST collection. Annotation of 534 unigenes was possible following the categories applied to Saccharomyces cerevisiae and the Universal Gene Ontology scheme. cDNAs were identified that encoded potential pathogenicity factors including four endopolygalacturonases, two exopolygalacturonases, and several metabolite transporters. The potential role of these genes, as well as those encoding signal transduction factors, in the infection process is discussed.

A convenient, Agrobacterium-mediated transient expression assay has been evaluated for rapid analysis of plant promoters and transcription factors in vivo. By simple infiltration of Agrobacterium cells carrying appropriate plasmid constructs into tobacco leaves in planta, reproducible expression assays could be conducted in as little as 2-3 days without using expensive equipment (e.g. biolistic gun or electroporation apparatus) or complicated procedures (e.g. preparation of protoplasts). Biotic and abiotic treatments could be applied to the intact plant to examine their influence on promoter activity and gene expression. Using this method, we have tested the stress-responsive as-1 and heat shock elements, yeast GAL4 transactivation system, two promoters of pathogenesis-related (PR) genes as well as a heat shock promoter. Through deletion analyses of tobacco PR1a promoter and a novel myb1 promoter, we have also successfully identified the cis-regulatory regions in these promoters that are responsive to salicylic acid treatment or tobacco mosaic virus infection. Together, our results demonstrate that Agrobacterium-mediated transient expression is a simple and efficient method for in vivo assays of plant promoters and transcription factors.