Transcriptome analysis of the fungal pathogen Rosellinia necatrix during infection of a susceptible avocado rootstock identifies potential mechanisms of pathogenesis.

BACKGROUND: White root rot disease caused by Rosellinia necatrix is one of the most important threats affecting avocado productivity in tropical and subtropical climates. Control of this disease is complex and nowadays, lies in the use of physical and chemical methods, although none have proven to be fully effective. Detailed understanding of the molecular mechanisms underlying white root rot disease has the potential of aiding future developments in disease resistance and management. In this regard, this study used RNA-Seq technology to compare the transcriptomic profiles of R. necatrix during infection of susceptible avocado 'Dusa' roots with that obtained from the fungus cultured in rich medium. RESULTS: The transcriptomes from three biological replicates of R. necatrix colonizing avocado roots (RGA) and R. necatrix growing on potato dextrose agar media (RGPDA) were analyzed using Illumina sequencing. A total of 12,104 transcripts were obtained, among which 1937 were differentially expressed genes (DEG), 137 exclusively expressed in RGA and 160 in RGPDA. During the root infection process, genes involved in the production of fungal toxins, detoxification and transport of toxic compounds, hormone biosynthesis, gene silencing and plant cell wall degradation were overexpressed. Interestingly, 24 out of the 137 contigs expressed only during R. necatrix growth on avocado roots, were predicted as candidate effector proteins (CEP) with a probability above 60%. The PHI (Pathogen Host Interaction) database revealed that three of the R. necatrix CEP showed homology with previously annotated effectors, already proven experimentally via pathogen-host interaction. CONCLUSIONS: The analysis of the full-length transcriptome of R. necatrix during the infection process is suggesting that the success of this fungus to infect roots of diverse crops might be attributed to the production of different compounds which, singly or in combination, interfere with defense or signaling mechanisms shared among distinct plant families. The transcriptome analysis of R. necatrix during the infection process provides useful information and facilitates further research to a more in -depth understanding of the biology and virulence of this emergent pathogen. In turn, this will make possible to evolve novel strategies for white root rot management in avocado.

GFP sheds light on the infection process of avocado roots by Rosellinia necatrix.

In order to monitor Rosellinia necatrix infection of avocado roots, we generated a plasmid vector (pCPXHY1eGFP) constitutively expressing EGFP and developed a protoplast transformation protocol. Using this protocol, four R. necatrix isolates were efficiently transformed and were shown to stably express EGFP homogeneously while not having any observable effect on pathogenicity. Confocal laser scanning microscopy (CLSM) images of avocado roots infected with the highly virulent isolate CH53-GFP demonstrated that fungal penetration of avocado roots occurs simultaneously at several random sites, but it occurs preferentially in the crown region as well as throughout the lenticels and in the junctions between epidermal cells. Not only were R. necatrix hyphae observed invading the epidermal and cortical root cells, but they were also able to penetrate the primary and secondary xylem. Scanning electron microscopy (SEM) images allowed detailed visualisation of the hyphal network generated by invasion of R. necatrix through the epidermal, cortical and vascular cells, including hyphal anastomosis and branching points. To our knowledge, this is the first report describing the construction of GFP-tagged strains belonging to the genus Rosellinia for monitoring white root rot using CLSM and SEM.

Mutations in the hepatocyte nuclear factor-1alpha/MODY3 gene in Japanese subjects with early- and late-onset NIDDM.

Recent studies have shown that mutations in the hepatocyte nuclear factor (HNF)-1alpha gene are the cause of maturity-onset diabetes of the young type 3 (MODY3). We have screened 193 unrelated Japanese subjects with NIDDM for mutations in this gene: 83 with early-onset NIDDM (diagnosis at <30 years of age) and 110 with late-onset NIDDM (diagnosis > or = 30 years of age). All of the members of the latter group also had at least one sibling with NIDDM. The 10 exons, flanking introns, and promoter region were amplified using polymerase chain reaction and were sequenced directly. Mutations were found in 7 of the 83 (8%) unrelated subjects with early-onset NIDDM. The mutations were each different and included four missense mutations (L12H, R131Q, K205Q, and R263C) and three frameshift mutations (P379fsdelCT, T392fsdelA, and L584S585fsinsTC). One of the 110 subjects with late-onset NIDDM was heterozygous for the missense mutation G191D. This subject, who was diagnosed with NIDDM at 64 years of age, also had a brother with NIDDM (age at diagnosis, 54 years) who carried the same mutation, suggesting that this mutation contributed to the development of NIDDM in these two siblings. None of these mutations were present in 50 unrelated subjects with normal glucose tolerance (100 normal chromosomes). Mutations in the HNF-1alpha gene occur in Japanese subjects with NIDDM and appear to be an important cause of early-onset NIDDM in this population. In addition, they are present in about 1% of subjects with late-onset NIDDM.

Comparison of nonradioactive cDNA probes for detection of potato spindle tuber viroid by dot-blot hybridization assay.

Six nonradioactive cDNA probes were compared for their sensitivities for detecting potato spindle tuber viroid (PSTVd) by dot-blot hybridization assay. Three biotinylated PSTVd cDNA probes, labeled by photoactivation with photobiotin, by nick translation or by random priming with biotinylated deoxyribonucleotides, were all capable of detecting 20 pg of purified PSTVd by a colorimetric assay and 2-20 pg by a chemiluminescent assay. Digoxigenin-labeled probe was able to detect 200 pg of purified PSTVd. Two biotinylated probes prepared with polymerase chain reaction (PCR) incorporating biotinylated dUTP or dATP were the most sensitive: 0.2-2 pg of PSTVd was detectable by both assays. All six probes could detect PSTVd also in extracts of infected tomato leaves at a dilution of up to 1/250-1/1250. These nonradioactive probes are equal to radioactive probes in their sensitivity, and the biotinylated probes produced with PCR amplification are particularly suitable for practical diagnosis, as they are sensitive and rapidly prepared in large quantities.

Development of diabetic nephropathy in Japanese patients with insulin-dependent diabetes mellitus: Tokyo Women's Medical College epidemiologic study.

To clarify the development of diabetic nephropathy in Japanese insulin-dependent diabetes mellitus (IDDM), 373 patients with IDDM who had no proteinuria at the first visit to our Diabetes Center were evaluated. The incidence of persistent proteinuria increased rapidly between 10 and 19 years of diabetes duration, and only a few new cases occurred thereafter. Patients whose ages at onset of IDDM were 9-17 years (n = 167) and 18-29 years (n = 90) had rapid development of persistent proteinuria compared to the 0 to 8-year group (n = 116) (p < 0.02 and p < 0.003, respectively). Females (n = 233) developed persistent proteinuria to a greater extent until 24 years of diabetes duration than males (n = 140) (p = 0.17). Development of proteinuria did not vary according to calendar year of diagnosis of IDDM or diabetes duration at the first visit. Renal insufficiency (serum creatinine of 2.0 mg/dL or greater) followed the onset of proteinuria; 60% of the patients with the onset of IDDM between 1951 and 1969 developed renal insufficiency 10 years after the onset of proteinuria, whereas 30% of patients with the onset of IDDM after 1970 developed this condition. Development of renal insufficiency did not vary according to sex, age at onset of IDDM, or age at onset of proteinuria. Renal failure requiring dialysis therapy occurred within 4 years after renal insufficiency. In conclusion, development of diabetic nephropathy in Japanese IDDM exhibits three stages. The onset of proteinuria appears to be influenced by age at onset of IDDM and sex.(ABSTRACT TRUNCATED AT 250 WORDS)

Infection Behavior of Venturia nashicola, the Cause of Scab on Asian Pears.

ABSTRACT The infection of Japanese pear by Venturia nashicola, the cause of scab on Asian pears (Japanese pear, Pyrus pylifolia var. culta; Chinese pear, P. ussuriensis), was examined using light and electron microscopy to determine the mechanism of resistance in pears. Early stages of infection were similar on the susceptible cv. Kosui, the resistant cv. Kinchaku, and the nonhost European pear (P. communis) cv. Flemish Beauty. V. nashicola penetrated only the cuticle layer on pear leaves and formed subcuticular hyphae on all three cultivars. Hyphae were localized in the pectin layer of pear leaves and never penetrated into the cytoplasm of epidermal cells. This restriction of fungal growth suggested that pectinases released by infection hyphae or subcuticular hyphae may be important in infection. Subcuticular hyphae were modified ultrastructurally in the pectin layer of resistant pear cultivars accompanied by fungal cell death. In contrast, fungal cells appeared intact in susceptible pear cultivars, suggesting the existence of resistance mechanisms.

A Reovirus Causes Hypovirulence of Rosellinia necatrix.

ABSTRACT White root rot, caused by Rosellinia necatrix, is a serious soilborne disease of fruit trees and other woody plants. R. necatrix isolate W370 contains 12 segments of double-stranded RNA (dsRNA) that is believed to represent a possible member of the family Reoviridae. W370 was weakly virulent and its hyphal-tip strains became dsRNA free and strongly virulent. The 12 segments of W370dsRNA were transmitted to hygromycin B-resistant strain RT37-1, derived from a dsRNA-free strain of W370 in all or none fashion through hyphal contact with W370. The W370dsRNA-transmitted strains were less virulent than their parent strain RT37-1 on apple seedlings, with mortality ranging between 0 to 16.7% in apple seedlings that were inoculated with the W370dsRNA-containing strains and 50 to 100% for seedlings inoculated with the dsRNA-free strains. Some W370dsRNA-containing strains killed greater than 16.7% of seedlings, but these were found to have lost the dsRNA in planta. These results indicate that W370dsRNA is a hypovirulence factor in R. necatrix. In addition, a strain lost one segment (S8) of W370dsRNA during subculture, and the S8-deficient mutant strain also exhibits hypovirulence in R. necatrix.

Effects of VIP and GRF on the release of growth hormone in perifused bovine adenohypophysis.

The effects of vasoactive intestinal polypeptide (VIP) and growth hormone releasing factor (GRF:hpGRF(1-29)-NH2) on the release of growth hormone (GH) from anterior pituitaries from cows were examined by using an in vitro superfusion system. The pituitaries were excised randomly from cycling cows, dissected to obtain medial portions, and minced to obtain cubes with approximate dimensions of 1.5mm on a side. For each perifusion setup, 5 pieces of pituitary tissues were chambered and flushed with modified KRB solution saturated with 95% O2-5% CO2 at 38C. Perifusion with media containing 10(-6) and 10(-7)M VIP for 30 min induced a significant release of GH during the treatments (P less than 0.05). VIP (10(-8)M) increased GH levels significantly (P less than 0.05), but to a minor degree. Perfusion with the media containing 10(-6), 10(-7) and 10(-8)M GRF for 30 min markedly increased the GH concentration and the effects continued up to 90 min after termination of the perifusion of the peptide (P less than 0.05, P less than 0.01). The GH releasing effects of GRF could be seen at doses as low as 10(-11)M GRF (P less than 0.05, P less than 0.01). These findings indicate that the GH releasing effect of VIP is less potent than that of GRF in cows.

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP), prostaglandin E2 (PGE2) and growth hormone releasing factor (GRF) on the release of growth hormone from cultured bovine anterior pituitary cells in vitro.

The effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on growth hormone (GH) release was compared with that of prostaglandin E2 (PGE2) and growth hormone releasing factor (GRF) from cultured bovine anterior pituitary cells in vitro. Both PACAP and PGE2 stimulated GH release at concentrations as low as 10(-9) and 10(-8) M, respectively, (P < 0.01). However, GRF released GH at a concentration as low as 10(-13) M (P < 0.01). Percent increases of GH compared with controls were not significantly different among GRF, PACAP, and PGE2 at 10(-7) M; however, the increases of GH by the 10(-8) M GRF, PACAP and PGE2 were 196, 118, and 27%, respectively, (P < 0.01), and 124, 65, and 1% in the 10(-9) M media, respectively, (P < 0.01). When GRF and somatostatin (SS) were added together, the GH releasing effect of GRF was blunted (P < 0.01). Similar bluntness were observed in PACAP and PGE2, when SS was added. The stimulatory effects of GRF and PGE2 together were similar to that by either GRF or PGE2 alone. When GRF and PACAP were added together, the GH released by both secretagogues was greater than that by PACAP alone (P < 0.01); however, a synergistic effect was not clear when compared with GRF alone. These findings suggest that PACAP and PGE2 may modulate the release of GH in cattle.

Effect of vasoactive intestinal peptide on the release of growth hormone in perifused pituitary and hypothalamus of the goat.

The effect of vasoactive intestinal peptide (VIP) on the release of growth hormone (GH) from the adenohypophysis of the goat was studied in vitro using the perifusion system. Two perifusion systems were employed to differentiate potential direct effects of VIP on the pituitary from indirect effects mediated through the hypothalamus. The first perifusion system used single chambers housing only pituitary fragments. The second system used two chambers in tandem, one containing hypothalamus and the second the pituitary fragments, the flow passing through the hypothalamic chamber first. VIP (10(-6), 10(-7), 10(-8)M) stimulated significant GH release in both perifusion systems (P less than 0.05, P less than 0.01) in a concentration related fashion. The quantity of GH release induced by the 10(-9)M and 10(-10)M VIP groups were not significant. Further, the GH released from the system that perifused the pituitary alone (10(-8)M VIP) was significantly higher than that containing both the hypothalamus and the pituitary fragments (P less than 0.05). The relative lower GH secretory response to the same dose of VIP applied to the hypothalamus-pituitary suggests that the perifused caprine hypothalamus may release an inhibitory factor, such as somatostatin in vitro. These results suggest that VIP stimulates GH release by acting directly on the adenohypophysis of the goat.

Microdialysis measurement of intracerebral somatostatin in the goat.

A microdialysis sampling technique for the intracerebral measurement of somatostatin (SS) in extracellular fluid was examined in the goat. The microdialysis probe (70-mm shaft, 0.5 mm outer diameter) contained at its tip a 4-mm length of copolymer dialysis membrane (20 kDa cut-off). Artificial cerebrospinal fluid (artificial CSF) was pumped through the probe tip at a rate of 4 microliters/min with a batter-driven syringe pump, and effluent fractions of dialysate (120 microliters) were collected every 30 min. An in vitro recovery test showed that changes in the SS concentration in dialysate were highly correlated (r = 0.95, P < 0.01) with those in the external medium, and the relative recovery averaged 2.0%. As a validation for in vivo microdialysis, trails were conducted with conscious behaving goats wherein the inflow dialysate was changed transiently from artificial CSF with low potassium (2.5 mM) to a solution of 300 mM KCl. Potassium-induced depolarization around the probe tip located in the preoptic area and in the hypothalamus induced an increase in SS concentrations in dialysate at each location. In the most remarkable response, the concentrations of SS were increased 6-fold and 11-fold in the first and second 30-min fractions, respectively, compared with prepotassium concentrations. These results suggest that intracerebral SS levels in extracellular fluid could be estimated from conscious behaving goats by the use of our intracerebral microdialysis system.

Effects of growth hormone (GH)-releasing hormone and its analogs on GH secretion from cultured adenohypophysial cells in cattle.

The effects of the human growth hormone (GH)-releasing hormone (hGRF(1-44)-NH2: hGRF-44), bovine GRF (bGRF(1-44)-NH2: bGRF-44), and their analogs (hGRF(1-29)-NH2, [D-Ala2]- hGRF(1-29)-NH2, bGRF(1-29)-NH2,[D-Ala2,Ala15]-bGRF(1-29)-NH2) as well as rat GRF(rGRF) on bovine GH release from anterior pituitary (AP) cells were studied in vitro in steers. The AP cells were incubated for 2 hr with the GRFs after preincubation for 3.5 d. Both of the hGRF-44 and hGRF(1-29)-NH2 (hGRF-29) significantly stimulated GH release from cultured cells at doses from 10(-14) to 10(-8)M (P < 0.01). The analog [D-Ala2]-hGRF-29 significantly induced GH release in media at doses from 10(-18) to 10(-8) M (P < 0.01). The bGRF-44, bGRF(1-29)-NH2 (bGRF-29), and [D-Ala2, Ala15]-bGRF-29 significantly induced GH release in media at doses as low as 10(-18), 10(-17), and 10(-16)M, respectively (P < 0.01). At doses from 10(-11) to 10(-8)M, there were no significant differences in GH-releasing potency between the hGRFs and bGRFs. The rGRF significantly stimulated GH release at doses ranging from 10(-14) to 10(-8) M (P < 0.01). The linear regression tests showed that the hGRFs, bGRFs, and rGRF, at doses from 10(-14) to 10(-8)M, induced GH release in a dose-related manner (P < 0.01). These results suggest that the hGRF, bGRF, and their analogs, as well as rGRF, are potent secretagogues of GH release from adenohypophysial cells in vitro in cattle.

Temporal bone findings in keratitis, ichthyosis, and deafness syndrome. Case report.

In 1981, the term KID syndrome was suggested for patients with congenital ichthyosis associated with deafness and keratitis. We had a chance to examine the temporal bone of an infant with this syndrome. This patient showed no auditory brain stem response in either ear. Temporal bone studies revealed cochleosaccular abnormality. These findings are offered as a possible explanation for the patient's deafness. The pathologic inner ear findings of congenital deafness syndromes associated with ichthyosis have been heretofore reported in Refsum's syndrome and in a case with universal alopecia. In these cases, the temporal bone pathologic findings were a result of cochleosaccular abnormality. From our case and previous reports, it is suggested that the deafness associated with congenital ichthyosis might be the result of cochleosaccular abnormality.

First Report of Tulip band breaking virus in Mosaic Diseased Tulip in Japan.

Tulip (Tulipa spp.) is an ornamental plant of major economic importance in Japan. Regions in Toyama Prefecture are some of the most productive for producing tulip bulbs, shipping approximately 50 million bulbs annually. However, mosaic diseases caused by viruses such as Tulip breaking virus (TBV) currently limit bulb production in these areas. Only the potyviruses TBV and Lily mottle virus (LMoV) have been reported infecting tulip in Japan. A virus isolate from tulip with flower-breaking symptom in Toyama Prefecture was tentatively named OE4 and was presumed to be LMoV after detection by LMoV-specific reverse transcription-polymerase chain reaction (RT-PCR) (4). When OE4 was mechanically inoculated on test plants (13 species from six families), RT-PCR confirmed that it infected plants in the Liliaceae (Tulipa spp., Lilium formosanum, and L. concolor) with mosaic symptoms but did not induce any symptoms in Chenopodium quinoa, Tetragonia tetragonoides, and Nicotiana benthamiana. According to Dekker et al. (2) LMoV and Tulip band breaking virus (TBBV) infected Tulipa spp. and TBBV did not infect C. quinoa, T. tetragonoides, N. clevelandii, and N. benthamiana, species that were local or systemic hosts for LMoV. To analyze the genomic sequence of OE4, a primer set was designed for amplifying the coat protein (CP) gene of LMoV, with 5' -GCAAATGAGACACTCAATG-3' as a forward primer and 5'-TTACATAGAAATTCCAAGTAAG-3' as a reverse primer. The fragment obtained was cloned and sequenced (DDBJ/EMBL/GenBank Accession No. AB078007). The CP gene of OE4 consisted of 825 nucleotides and had 86.5% identity (90.6% identity in deduced amino acid sequence) with the CP gene of LMoV-Netherlands (S44147) (3). When it was compared with partial sequences (277 nucleotides) of LMoV (S60810) and TBBV (S60805) (2), the nucleotide sequence identities were 89.9 and 96.4%, respectively. Multiple alignment and a phylogenetic tree based on the 277 nt of the tulip potyviruses, including OE4, showed the close relationship between OE4 and TBBV. These results indicated that OE4 was an isolate of TBBV. However, the CP amino acid sequence identity between TBBV and LMoV was more than 80%, and it seemed logical that TBBV be assigned to a strain of LMoV, according to potyvirus species demarcating criteria (1). Another screening using RT-PCR based detection, and an inoculation test for 55 flower-breaking tulips collected from fields in Toyama Prefecture revealed that four tulip plants were infected with TBBV and 54 with TBV. This suggests that TBV is more prevalent than TBBV, and we need to produce an attenuated virus of TBV, which will be effective for managing tulip breaking disease in the prefecture. References: (1) P. H. Berger et al. Family Potyviridae. Pages 703-724 in: Virus Taxonomy. Academic Press, San Diego, CA, 2000. (2) E. L. Dekker et al. J. Gen. Virol. 74:881, 1993. (3) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991. (4) T. Se and S. Kanematsu. Ann. Phytopathol. Soc. Jpn. 64:420, 1998.

Serum parathyroid hormone and calcitonin levels in racehorses with fracture.

Serum parathyroid hormone (PTH) and calcitonin (CT) levels in fractured racehorses were measured by radioimmunoassay. Racehorses with fracture of large bone such as the radius, third metacarpus, third carpus, digital bone or tibia, showed normal PTH level and elevated CT level in the serum. Serum PTH level was slightly higher in racehorses with sesamoid bone fracture compared to that of healthy racehorses, but not statistically significant. Moreover, serum CT level of racehorses with sesamoid bone fracture was significantly higher than that of healthy racehorses. Racehorses with sesamoid bone fracture and large bone fracture might be in different conditions of calcium regulation.

Gastric tumor from metastasis of breast cancer.

Metastatic tumours of the stomach have been reported to result from various types of cancer. Among them, gastric metastasis from breast cancer has been recognised in 0.3-18% patients (1-4). Here, a rare case of metastatic gastric tumour derived from breast carcinoma is reported. Gastric endoscopy confirmed a large, friable mass (approximately 5 cm in diameter) in the upper part of the gastric body. The mass within the stomach was difficult to distinguish from primary gastric cancer, although biopsies of this lesion revealed the characteristics of adenocarcinoma. In addition, immunohistochemistry showed the positive expression of mammaglobin. Taken together, the evidence pointed to metastasis of breast cancer to the stomach. The patient was treated with hormonal therapy (letrozole), and the size of the metastasis in the stomach was markedly reduced. Therefore, a gastric metastasis from breast cancer was diagnosed successfully using immunohistochemistry and unnecessary surgery was avoided. In conclusion, although gastric metastatic tumours derived from breast carcinoma are rare, their accurate pre-operative diagnosis and appropriate systemic treatment is essential.