Characterization of a PR-10 pathogenesis-related gene family induced in rice during infection with Magnaporthe grisea.

A partial cDNA with homology to the PR-10 class of pathogenesis-related proteins was used to screen a rice genomic library. One 16-kb genomic clone contained three genes with PR-10 similarity. These genes, RPR10a, RPR10b, and RPR10c, were arranged in tandem and separated by approximately 2.5 kb. RPR10a cDNA was obtained by reverse transcription-polymerase chain reaction, and sequence analysis revealed that RPR10a and RPR10b encode predicted proteins of 158 and 160 amino acids, respectively, and share 71% amino acid identity. RPR10c appears to be a nonfunctional pseudogene. Gene-specific probes were used to study transcript accumulations of the three RPR10 genes in rice plants following inoculation with Magnaporthe grisea. RPR10a transcripts were induced from a low basal level within 12 h after inoculation and showed a second higher level induction at 48 h, which continued throughout the 144 h it was examined. In addition, RPR10a was induced strongly by salicylic and jasmonic acid applications to rice plants. Transcripts of RPR10b also were enhanced by M. grisea, but were not strongly visible until 48 h after inoculation. Tissue prints of M. grisea-infected rice leaves when the RPR10a-specific probe was used indicate that RPR10a is expressed most strongly in a localized fashion in response to the pathogen.

Vascular defense responses in rice: peroxidase accumulation in xylem parenchyma cells and xylem wall thickening.

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae is a vascular pathogen that elicits a defensive response through interaction with metabolically active rice cells. In leaves of 12-day-old rice seedlings, the exposed pit membrane separating the xylem lumen from the associated parenchyma cells allows contact with bacterial cells. During resistant responses, the xylem secondary walls thicken within 48 h and the pit diameter decreases, effectively reducing the area of pit membrane exposed for access by bacteria. In susceptible interactions and mock-inoculated controls, the xylem walls do not thicken within 48 h. Xylem secondary wall thickening is developmental and, in untreated 65-day-old rice plants, the size of the pit also is reduced. Activity and accumulation of a secreted cationic peroxidase, PO-C1, were previously shown to increase in xylem vessel walls and lumen. Peptide-specific antibodies and immunogold-labeling were used to demonstrate that PO-C1 is produced in the xylem parenchyma and secreted to the xylem lumen and walls. The timing of the accumulation is consistent with vessel secondary wall thickening. The PO-C1 gene is distinct but shares a high level of similarity with previously cloned pathogen-induced peroxidases in rice. PO-C1 gene expression was induced as early as 12 h during resistant interactions and peaked between 18 and 24 h after inoculation. Expression during susceptible interactions was lower than that observed in resistant interactions and was undetectable after infiltration with water, after mechanical wounding, or in mature leaves. These data are consistent with a role for vessel secondary wall thickening and peroxidase PO-C1 accumulation in the defense response in rice to X. oryzae pv. oryzae.

Acute spiral ganglion lesions change the tuning and tonotopic organization of cat inferior colliculus neurons.

Many studies have reported plastic changes in central auditory frequency organization after chronic cochlear lesions. These studies employed mechanical, acoustic or drug-induced disruptions of restricted regions of the organ of Corti that permanently alter its tuning and sensitivity and require an extended recovery period before central effects can be measured. In this study, mechanical lesions were made to 1 mm sectors of the spiral ganglion (SG). These lesions remove a restricted portion of the cochlear output, but leave the organ of Corti and basilar membrane intact. Multiunit mapping assessed the pre- and post-lesion tonotopic organization of the inferior colliculus (IC). Immediately after SG lesions, IC neurons previously tuned to the lesion frequencies became less sensitive to those frequencies but more sensitive to lesion edge frequencies, resulting in a shift in their characteristic frequencies (CFs). Notches in the excitatory response areas at frequencies corresponding to the lesion frequencies and expansion of spatial tuning curves were also observed. CFs of neurons tuned to unlesioned frequencies were unchanged. These results suggest that 'plastic' changes similar to those observed after long survival times in previous studies require little or no experience and occur within minutes to hours following the lesion.

Human papillomavirus status in the prediction of high-grade cervical intraepithelial neoplasia in patients with persistent low-grade cervical cytological abnormalities.

The role of human papillomavirus (HPV) detection in the management of patients with persistent low-grade (mild dyskaryosis or less) cervical cytological abnormalities is unclear. We have analysed cytological material from 167 such patients both cytologically and by non-isotopic in situ hybridisation (NISH) for HPV 16, 18, 31 and 33 and consensus primer polymerase chain reaction (PCR) amplification followed by both generic and specific typing for these HPV types. Cervical intraepithelial neoplasia (CIN) 2 or 3 was present in 40 of 167 patients (23.9%), and the positive predictive values (PPVs) for the presence of CIN 2 or 3, of moderate or severe dyskaryosis at repeat cytology and an HPV-positive NISH and generic PCR signal were 100%, 66% and 42% respectively. The corresponding sensitivities were 48%, 68% and 87%. Addition of cytology to molecular analysis improved both PPV and sensitivity, the best combination being NISH and cytopathology (PPV 71%, sensitivity 87%). These data demonstrate that the presence of CIN 2 or 3 in patients with mild cytological abnormalities can be predicted by molecular detection of HPV in some cases, particularly when combined with cytological analysis. However, the magnitude of this prediction is dependent on the population of patients studied, and the clinical role of this approach therefore remains to be defined.

Homologous recombination between plasmid DNA molecules in maize protoplasts.

The requirements for homologous recombination between plasmid DNA molecules have been studied using the PEG (polyethylene glycol)-mediated transformation system of maize (Zea mays L.) protoplasts coupled with the transient expression assay for beta-glucuronidase (GUS). Two plasmids were introduced into maize protoplasts; one plasmid (pB x 26) contained a genomic clone of the Adh1 maize gene; the other plasmid (piGUS) was a promoterless construction containing part of intron A of the Adh1 gene fused to the gusA coding sequence. Thus, the two vectors shared an effective homologous region consisting of a 459 bp (HindIII-PvuII) fragment of the Adh1 intron A sequence. An active gusA fusion gene would result upon homologous recombination between the plasmids within the intron A sequence, and indeed GUS activity was observed in extracts following co-transformation of maize protoplasts with the two plasmids. The presence of recombinant DNA molecules in protoplast DNA isolated 1 day after co-transformation was verified using polymerase chain reactions (PCR) and Southern blots. For efficient homologous recombination, both plasmids had to be linearized. The recombination reaction was induced by restriction of the plasmid molecules either inside the effective homologous region or at the borders of the intron sequence. However, the presence of even small, terminal, nonhomologous sequences at the 3' end of the pB x 26 fragment inhibited the recombination reaction. Also, both ends of the linearized piGUS DNA molecules were involved in the recombination reaction. The results revealed some features of homologous recombination reactions occurring in plant cells which cannot be accommodated by mechanisms postulated for similar reactions in animal system and in lower eukaryotes.