Host-root exudates increase gene expression of asparagine synthetase in the roots of a hemiparasitic plant Triphysaria versicolor (Scrophulariaceae).

Triphysaria is a facultative root parasite in the Scrophulariaceae family. Similar to other related parasites, the development of the parasitic life cycle is initiated by molecular signals released from appropriate host roots. Using a differential display, we isolated cDNAs preferentially abundant in T. versicolor roots exposed to Trifolium repens (white clover) root exudates in vitro. Sequence analysis indicated that one of the differentially expressed cDNAs had significant homology to the nitrogen-assimilating enzyme, asparagine synthetase (AS). T. versicolor AS cDNA clones were isolated and placed into three distinct classes on the basis of nucleotide sequence variations. All three classes encoded identical AS proteins. AS was expressed in both roots and shoots of in-vitro-cultured T. versicolor. Steady-state levels of AS mRNA increased in T. versicolor roots several-fold when seedlings were exposed to exudate obtained from hydroponically grown Arabidopsis thaliana roots. Therefore, AS transcript levels increased in response to exudates from two different hosts (Trifolium and Arabidopsis). The T. versicolor AS message levels increased to a similar magnitude when seedlings were incubated in the dark. Interestingly, AS levels were unaffected by treatment with the Striga haustoria inducer 2,6-dimethoxybenzoquinone. The potential role of AS in root parasitism is discussed.

In vivo transfer of barley stripe mosaic hordeivirus ribonucleotides to the 5' terminus of maize stripe tenuivirus RNAs.

The Tenuivirus maize stripe virus (MStV) shares many properties with viruses in the genus Phlebovirus of the family Bunyaviridae. Besides genome organization and gene expression strategies, one property shared by these plant- and vertebrate-infecting viruses is that transcription gives rise to virus-specific mRNAs containing nonviral 5'-terminal nucleotide sequences. The 5'-terminal nucleotides are believed to be derived from host mRNA sequences as a result of "cap-snatching." We investigated whether specific nucleotide sequences could serve as primer donors for cap-snatching in vivo. Barley (Hordeum vulgare) plants were singly and doubly infected with MStV and the Hordeivirus barley stripe mosaic virus (BSMV). A reverse transcription-PCR assay was used to identify chimeric BSMV/MStV RNAs. Specific reverse transcription-PCR products were detected from doubly infected plants by using one PCR primer corresponding to the 5' termini of the BSMV RNAs (alpha, beta, and gamma) and a second primer complementary to MStV RNA 4. The resulting cDNAs were cloned, and nucleotide sequence analysis showed them to be chimeric, containing BSMV 5'-terminal sequences as well as MStV RNA 4 sequences. All clones contained the BSMV RNA 5' primer nucleotide sequence, but they also showed characteristics common to Tenuivirus mRNAs. More than 80% of the clones contained BSMV RNA nucleotides not present on the PCR primer. Several lacked the exact 5' terminus of MStV RNA 4, a feature also seen for viruses in the Bunyaviridae. These data show that heterologous virus RNAs (BSMV) can serve as primer donors for MStV mRNA capped RNA-primed transcription in doubly infected plants.

A Small RNA Resembling the Beet Western Yellows Luteovirus ST9-Associated RNA Is a Component of the California Carrot Motley Dwarf Complex.

ABSTRACT Virions were purified from Anthriscus cerefolium or Coriandrum sativum plants infected with the viruses that cause California carrot motley dwarf. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virion preparations yielded a single prominent protein species of approximately 28,000 molecular weight; however, denaturing agarose gel electrophoresis showed that virions contained three prominent single-stranded RNAs of approximately 5.6, 4.2, and 2.8 kb. Northern hybridization analyses, using transcripts generated from cloned cDNAs that corresponded to each of the virion RNAs, showed that the 5.6- and 4.2-kb RNAs were the genomic RNAs of the carrot red leaf luteovirus (CRLV) and the carrot mottle umbravirus (CMoV), respectively. Virions also contained an approximately 1.3-kb RNA related to the CMoV genomic RNA. The 2.8-kb RNA did not hybridize with CRLV or CMoV cRNA probes. Analysis of naturally infected carrot (Daucus carota) plants showed that CRLV, CMoV, and the 2.8-kb RNA were always present in carrot motley dwarf-affected plants. Greenhouse aphid- and mechanical-transmission experiments showed that the 2.8-kb RNA was consistently present in plants also infected by both CRLV and CMoV, but never in plants infected by only CMoV. Near full-length cloned cDNAs corresponding to the 2.8-kb RNA were prepared, and the complete nucleotide sequence was determined to be 2,835 nucleotides. Two large open reading frames (ORFs), 1a and 1b, were present within the sequence and were separated by an amber (UAG) stop codon. A third ORF (ORF 2), capable of encoding a protein of 4,289 molecular weight, was located near the 3' terminus. BLASTP results showed that the 2.8-kb RNA was most closely related to the beet western yellows luteovirus (BWYV) ST9-associated RNA. Based on its biological and molecular characteristics, we have named the 2.8-kb RNA the CRLV-associated RNA (CRLVaRNA).

Maize stripe tenuivirus RNA2 transcripts in plant and insect hosts and analysis of pvc2, a protein similar to the Phlebovirus virion membrane glycoproteins.

The complete sequence of the maize stripe tenuivirus (MStV) RNA2 was determined (3337 nucleotides). RNA2 contains two large open reading frames (ORFs) arranged in an ambisense orientation and specific RNAs of ca. 700 and 2600 nucleotides corresponding to the ORFs were detected in MStV-infected plants and planthoppers. The deduced amino acid sequence of the 23,500 MW protein (pv2) encoded by viral RNA2 (vRNA2) was similar to proteins encoded by the rice stripe (RStV) and rice hoja blanca tenuiviruses vRNA2. Sequence analysis suggested that pv2 is membrane associated. The 93,900 MW protein (pvc2) encoded by viral complementary MStV RNA2 (vcRNA2) was similar to the 94,000 MW protein of RStV RNA2 and to the virion membrane glycoproteins for Phlebovirus members of the Bunyaviridae. The phlebovirus glycoprotein cleavage site was similar to a region in the MStV and RStV proteins suggesting that the tenuivirus pvc2 may be processed analogous to the phlebovirus glycoproteins.

Predicted probabilities of hospital death as a measure of admission severity of illness.

This paper evaluates a new method for assessing hospital admission severity of illness based on disease-specific models (logistic regression) of the probability of in-hospital mortality. Results for the 26 disease groups in MDC 4--Diseases of the Respiratory System, MDC 5--Diseases of the Circulatory System, and MDC 6--Diseases of the Digestive System are presented using data on all 1991 admissions from 111 hospitals throughout the United States. These disease models are empirically derived using clinical findings from laboratory, radiology, pathology, diagnostic procedures, patient history and physical exam, as well as patient age and sex. Each predictive algorithm is presented, and the strong predictive performance of these models is indicated by the average C statistic of .870. A predicted probability of death is calculated for each hospital patient in the study sample, and these probabilities comprise a continuous variable that indicates admission severity of illness.

Differential expression of phenylalanine ammonia-lyase and chalcone synthase during soybean nodule development.

We have used conserved and nonconserved regions of cDNA clones for phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) isolated from a soybean-nodule cDNA library to monitor the expression of members of the two gene families during the early stages of the soybean-Bradyrhizobium japonicum symbiosis. Our results demonstrate that subsets of the PAL and CHS gene families are specifically induced in soybean roots after infection with B. japonicum. Furthermore, by analyzing a supernodulating mutant line of soybean that differs from the wild-type parent in the number of successful infections, we show that the induction of PAL and CHS is related to postinfection events. Nodulated roots formed by a Nod+ Fix- strain of B. japonicum, resembling a pathogenic association, display induction of another distinct set of PAL and CHS genes. Our results suggest that the symbiosis-specific PAL and CHS genes in soybean are not induced by stress or pathogen interaction.