Predictors of Quality of Life Decline in Patients with Oligometastases treated with Stereotactic Ablative Radiotherapy: Analysis of the Population-Based SABR-5 Phase II Trial.

AIMS: Most patients experience stable quality of life (QoL) after stereotactic ablative radiotherapy (SABR) treatment for oligometastases. However, a subset of patients experience clinically relevant declines in QoL on post-treatment follow-up. This study aimed to identify risk factors for QoL decline. MATERIALS AND METHODS: The SABR-5 trial was a population-based single-arm phase II study of SABR to up to five sites of oligometastases. Prospective QoL was measured using treatment site-specific tools at pre-treatment baseline and 3, 6, 9, 12, 15, 18, 21, 24, 30 and 36 months after treatment. The time to persistent QoL decline was calculated as the time from SABR to the first decline in QoL score meeting minimum clinically important difference with no improvement to baseline score on subsequent assessments. Univariable and multivariable logistic regression analyses were carried out to determine factors associated with QoL decline. RESULTS: One hundred and thirty-three patients were included with a median follow-up of 32 months (interquartile range 25-43). Thirty-five patients (26%) experienced a persistent decline in QoL. The median time until persistent QoL decline was not reached. The cumulative incidence of QoL decline at 2 and 3 years were 22% (95% confidence interval 14.0-29.6) and 40% (95% confidence interval 28.0-51.2), respectively. In multivariable analysis, disease progression (odds ratio 5.23, 95% confidence interval 1.59-17.47, P = 0.007) and adrenal metastases (odds ratio 9.70, 95% confidence interval 1.41-66.93, P = 0.021) were associated with a higher risk of QoL decline. Grade 3 or higher (odds ratio 3.88, 95% confidence interval 0.92-16.31, P = 0.064) and grade 2 or higher SABR-associated toxicity (odds ratio 2.24, 95% confidence interval 0.85-5.91, P = 0.10) were associated with an increased risk of QoL decline but did not reach statistical significance. CONCLUSIONS: Disease progression and adrenal lesion site were associated with persistent QoL decline following SABR. The development of grade 3 or higher toxicities was also associated with an increased risk, albeit not statistically significant. Further studies are needed, focusing on the QoL impact of metastasis-directed therapies.

Prospective Longitudinal Assessment of Quality of Life After Stereotactic Ablative Radiotherapy for Oligometastases: Analysis of the Population-based SABR-5 Phase II Trial.

AIMS: To evaluate longitudinal patient-reported quality of life (QoL) in patients treated with stereotactic ablative radiotherapy (SABR) for oligometastases. MATERIALS AND METHODS: The SABR-5 trial was a population-based single-arm phase II study of SABR to up to five sites of oligometastases, conducted in six regional cancer centres in British Columbia, Canada from 2016 to 2020. Prospective QoL was measured using treatment site-specific QoL questionnaires at pre-treatment baseline and at 3, 6, 9, 12, 15, 18, 21, 24, 30 and 36 months after treatment. Patients with bone metastases were assessed with the Brief Pain Inventory (BPI). Patients with liver, adrenal and abdominopelvic lymph node metastases were assessed with the Functional Assessment of Chronic Illness Therapy-Abdominal Discomfort (FACIT-AD). Patients with lung and intrathoracic lymph node metastases were assessed with the Prospective Outcomes and Support Initiative (POSI) lung questionnaire. The two one-sided test procedure was used to assess equivalence between the worst QoL score and the baseline score of individual patients. The mean QoL at all time points was used to determine the trajectory of QoL response after SABR. The proportion of patients with 'stable', 'improved' or 'worsened' QoL was determined for all time points based on standard minimal clinically important differences (MCID; BPI worst pain = 2, BPI functional interference score [FIS] = 0.5, FACIT-AD Trial Outcome Index [TOI] = 8, POSI = 3). RESULTS: All enrolled patients with baseline QoL assessment and at least one follow-up assessment were analysed (n = 133). On equivalence testing, the patients' worst QoL scores were clinically different from baseline scores and met MCID (BPI worst pain mean difference: 1.8, 90% confidence interval 1.19 to 2.42]; BPI FIS mean difference: 1.68, 90% confidence interval 1.15 to 2.21; FACIT-AD TOI mean difference: -8.76, 90% confidence interval -11.29 to -6.24; POSI mean difference: -4.61, 90% confidence interval -6.09 to -3.14). However, the mean FIS transiently worsened at 9, 18 and 21 months but eventually returned to stable levels. The mean FACIT and POSI scores also worsened at 36 months, albeit with a limited number of responses (n = 4 and 8, respectively). Most patients reported stable QoL at all time points (range: BPI worst pain 71-82%, BPI FIS 45-78%, FACIT-AD TOI 50-100%, POSI 25-73%). Clinically significant stability, worsening and improvement were seen in 70%/13%/18% of patients at 3 months, 53%/28%/19% at 18 months and 63%/25%/13% at 36 months. CONCLUSIONS: Transient decreases in QoL that met MCID were seen between patients' worst QoL scores and baseline scores. However, most patients experienced stable QoL relative to pre-treatment levels on long-term follow-up. Further studies are needed to characterise patients at greatest risk for decreased QoL.

Validation of the Prognostic Utility of ESTRO/EORTC Oligometastatic Disease Classification: A Secondary Analysis From the Population-Based Phase II SABR-5 Trial.

PURPOSE: The recently developed European Society for Radiotherapy and Oncology (ESTRO)/European Organization for Research and Treatment of Cancer (EORTC) oligometastatic disease (OMD) classification has not been validated in terms of its prognostic significance. This study stratified patients from the phase II SABR-5 trial based on ESTRO/EORTC criteria and compared progression-free survival (PFS) and overall survival (OS) to determine the prognostic significance of the classification scheme. METHODS AND MATERIALS: The SABR-5 trial was a single arm phase II study conducted at the 6 regional cancer centers across British Columbia (BC), Canada, where SABR for oligometastases was only offered on trial. Patients with up to 5 oligometastases (total or not controlled by prior treatment and including induced OMD) underwent SABR to all lesions. Patients were 18 years of age or older, Eastern Cooperative Oncology Group 0 to 2, and life expectancy ≥6 months. PFS and OS were calculated using the Kaplan-Meier method and differences between OMD groups were assessed with log-rank tests. Univariable and multivariable analyses were performed using Cox regression modeling. RESULTS: Between November 2016 and July 2020, 381 patients underwent SABR on trial. Median follow-up was 27 months (interquartile range, 18-36). The most frequent OMD group was de novo OMD (69%), followed by repeat (16%) and induced (13%). OMD groups differed significantly in PFS (P < .001) but not OS (P = .069). The OMD classification was an independent predictor of both PFS (P = .005) and OS (P = .002). Of the 5 classification factors, only chronicity (synchronous, hazard ratio, 0.52; P = .027) and oligoprogression (hazard ratio, 2.05; P = .004) were independently prognostic for OS. CONCLUSIONS: In this large prospective cohort, the ESTRO/EORTC classification was an independent predictor of PFS and OS and should be used to identify specific patient groups for clinical trials. In this trial population, the prognostic power is largely attributable to chronicity and oligoprogression. Simplification of the framework may be possible in the future and allow for greater ease of use; however, further data on underrepresented OMD groups and histologies will be required.

The OsIME4 gene identified as a key to meiosis initiation by RNA in situ hybridization.

The formation of asexual seeds in plants holds great promise as a breeding system for one-line hybrid rice. Entry into meiosis is a key developmental decision in gametogenesis, especially in formation of asexual seeds in plants. Apomeiosis in MeMCs can be achieved by identifying and manipulating meiosis-specific genes. Using methods based on in situ hybridization and expression analysis, we identified OsIME4 (inducer of meiosis 4) sense and antisense transcripts involved in rice meiosis initiation, similar to initiation of meiosis in budding yeast. Our data suggest that the OsIME4 sense transcript, which encodes a putative mRNA N6-adenosine methyltransferase, keeps rice cells at mitosis stage through some form of epigenesis (DNA/RNA methylation), and the non-coding antisense transcript of OsIME4 converts the cell status from mitosis to meiosis by inhibiting expression (transcription and translation) of the sense transcript. We identified that the non-coding antisense transcript of OsIME4 converts archesporial cell status from mitosis to meiosis by inhibiting expression of the OsIME4 sense transcript in rice. Our results provide novel insights into meiosis initiation in rice and for engineering of apomixis in sexual crops by manipulating the OsIME4 sense and antisense transcripts, which has great promise for producing apomictic rice in the future.

Whole Brain Radiotherapy Versus Stereotactic Radiosurgery in Poor-Prognosis Patients with One to 10 Brain Metastases: A Randomised Feasibility Study.

AIMS: A significant proportion of patients with brain metastases have a poor prognosis, with a life expectancy of 3-6 months. To determine the optimal radiotherapeutic strategy for brain metastases in this population, we conducted a randomised feasibility study of whole brain radiotherapy (WBRT) versus stereotactic radiosurgery (SRS). MATERIALS AND METHODS: Patients with a life expectancy of 3-6 months and between one and 10 brain metastases with a diameter ≤4 cm were enrolled at six Canadian cancer centres. Patients were randomly assigned (1:1) to receive either WBRT (20 Gy in five fractions) or SRS (15 Gy in one fraction). The primary end point was the rate of accrual per month. Secondary feasibility and clinical end points included the ratio of accrued subjects to screened subjects. This trial is registered with ClinicalTrials.gov (number NCT02220491). RESULTS: In total, 210 patients were screened to enrol 22 patients into the trial; 20 patients were randomised between the two arms. Two patients did not receive treatment because one patient died and another patient withdrew consent after being enrolled. Patients were accrued between January 2015 and November 2017; the accrual rate was 0.63 patients/month. The most common reasons for exclusion were anticipated median survival outside the required range (n = 40), baseline Karnofsky Performance Score below 70 (n = 28) and more than 10 brain metastases (n = 28). The median follow-up was 7.0 months and the median survival was 7.0 months for all patients in the trial. The median intracranial progression-free survival was 1.8 months in the SRS arm and 9.2 months in the WBRT arm. There were five grade 3+ toxicities in the SRS arm and one grade 3+ toxicity in the WBRT arm; no grade 5 toxicities were observed. The cumulative rates of retreatment were 40% in the SRS arm and 40% in the WBRT arm. CONCLUSIONS: A randomised trial evaluating WBRT versus SRS in patients with one to 10 metastases and a poor prognosis is feasible. A slower than expected accrual rate and difficulties with accurate prognostication were identified as issues in this feasibility study. A larger phase III randomised trial is planned to determine the optimal treatment in this patient population.

Effects of Beet necrotic yellow vein virus in Spinach Cultivars.

Beet necrotic yellow vein virus (BNYVV) causes one of the most economically destructive diseases of sugar beet (Beta vulgaris), rhizomania, which may reduce sugar yield by 80%. This field investigation was conducted to evaluate the interactions between spinach genotypes and different BNYVV strains, and to determine whether BNYVV is transmitted through spinach seeds. Eight commercial spinach cultivars were planted in two BNYVV-infested fields and two control fields in Salinas, CA in 2009. Spinach plants in the BNYVV-infested fields showed disease symptoms of yellow-green or light-green vein clearing, mottling, or yellow-green chlorotic lesions on younger leaves as early as 28 days after planting (four- to six-true leaf stage). Leaves may also become stiff, more crinkled, and necrotic. There was an increase of lateral roots and leaf number but a decrease in leaf weight compared to healthy plants. Infected plants often became stunted, deformed, wilted, and dead. Symptomatic leaves and roots from plants with or without leaf symptoms in BNYVV-infested fields all tested positive for BNYVV by enzyme-linked immunosorbent assay. A more aggressive (resistance-breaking) strain of BNYVV led to higher disease incidence in spinach than in the wild type. BNYVV was not transmitted through spinach seeds. There were significant differences in disease development among cultivars, with disease incidence ranging from 8 to 44%, suggesting that genetic improvement of BNYVV resistance through spinach breeding should be feasible.

Radiation oncology in a Canadian province: measures of workload and treatment complexity.

AIMS: A growing and aging population is associated with an increased incidence of cancer. Advances in radiotherapy technology have changed the way radiation is planned and delivered. This population-based study documented changes in workload and treatment complexity over a 10 year period in a Canadian province. MATERIALS AND METHODS: We examined the population-based radiation records of a provincial Canadian cancer centre from 2000 (or from 2005 for some measures) to 2009 inclusive. We propose new measures of workload and treatment complexity currently used in our centre that can be easily adopted by other cancer centres. RESULTS: Workload measured by total new-to-doctor consultations increased 30% from 2000 to 2009 (3.3% annually, P=0.0008). Total treatment commencements increased 35% over the same time (3.9% annually, P<0.0001) but linear accelerator (linac) commencements increased at a slower rate of 2.0% annually (P=0.0002). The rates of increase in consultations and total commencements were faster than the rates of increase in the total population, the population over age 50 years, or the incidence of cancer. Implementation of stereotactic radiosurgery and increased brachytherapy treatments explain much of the increased workload. Measures of treatment complexity including simulations per linac course and radiation computer plans per linac course increased at steady rates of 3.6% (P=0.0019) and 3.2% (P=0.0088) annually, respectively, but portals (fields) per linac treatment course increased exponentially after the implementation of intensity-modulated radiotherapy. The number of fractions per linac patient declined by 2.6% annually (P<0.0001). CONCLUSIONS: This population-based study showed that radiation oncology workload increased at faster rates than the population or incidence of cancer. Measures of treatment complexity indicate an increasing investment for each course of linac treatment, but also the adoption of hypofractionated regimens. These results indicate that radiotherapy manpower requirements cannot be based on population or cancer incidence alone if current technological trends continue.

Impaired mitochondrial oxidative phosphorylation in multiple insulin-sensitive tissues of humans with type 2 diabetes mellitus.

Alteration of mitochondrial oxidative phosphorylation (OXPHOS) may contribute to insulin resistance. It is unclear, however, which characteristics are common to insulin-sensitive tissues. Using an oligonucleotide microarray and quantitative real-time polymerase chain reaction, the gene expression profiles of skeletal muscle, visceral adipose tissue and liver from autopsy donors with and without type 2 diabetes mellitus were determined. Common dysregulated genes were enriched in mitochondrial OXPHOS, and most of these genes were down-regulated in both the skeletal muscle and visceral adipose tissue of diabetic subjects, but up-regulated in diabetic liver. Messenger RNA (mRNA) for peroxisome proliferator-activated receptor-gamma co-activator 1alpha was significantly increased in diabetic liver but significantly reduced in diabetic skeletal muscle. Tumour necrosis factor-alpha mRNA was significantly down-regulated in diabetic visceral adipose tissue. The mitochondrial DNA content was slightly, though not significantly, reduced in diabetic liver and diabetic skeletal muscle. It is concluded that defects in OXPHOS genes and individual transcription co-factors in insulin-sensitive tissues may play an important role in the development of type 2 diabetes and the insulin-resistant state.

First Report of Beet necrotic yellow vein virus Infecting Spinach in California.

In 2009, plants from two spinach (Spinacia oleracea) experimental fields in Monterey County and one commercial spinach field in Ventura County of California exhibited vein-clearing, mottling, interveinal yellowing, and stunting symptoms. For experimental fields, up to 44% of spinach plants have symptoms. With a transmission electron microscope, rigid rod-shaped particles with central canals were observed from plant sap of the symptomatic spinach. Analysis with a double-antibody sandwich-ELISA assay for Beet necrotic yellow vein virus (BNYVV) showed that all 10 symptomatic plants we tested were positive and 5 asymptomatic plants were negative. Symptomatic spinach from both counties was used for mechanical transmission experiments. Chenopodium quinoa, Tetragonia expansa, and Beta vulgaris (sugar beet) showed chlorotic local lesions and B. macrocarpa and spinach showed vein-clearing, mottling, and systemic infections. To further confirm the presence of BNYVV, reverse transcription (RT)-PCR was conducted. Total RNA was extracted from field- and mechanically inoculated symptomatic spinach plants using an RNeasy Plant Kit (Qiagen Inc., Valencia, CA) and used as a template in RT-PCR. Forward and reverse primers specific to the BNYVV RNA-3 P25 protein gene from the beet isolate were used (2). Amplicons of the expected size (approximately 860 bp) were obtained. Four RT-PCR products were sequenced and the sequences were identical (GenBank Accession No. GU135626). Sequences from the spinach plants had 97 to 99% nucleotide and 94 to 100% amino acid identity with BNYVV RNA-3 P25 protein sequences available in the GenBank. On the basis of the data from electron microscopy, indicator plants, serology, and cDNA sequencing, the virus was identified as BNYVV. BNYVV has been reported from spinach fields in Italy (1). To our knowledge, this is the first report of BNYVV occurring naturally on spinach in California. Since BNYVV is transmitted by the zoospores of the soil-inhabiting plasmodiophorid Polymyxa betae, it could be a new threat to spinach production in the state. References: (1) C. R. Autonell et al. Inf. Fitopatol. 45:43, 1995. (2) H.-Y. Liu and R. T. Lewellen, Plant Dis. 91:847, 2007.

Spinach (Spinacia oleracea) is a New Natural Host of Impatiens necrotic spot virus in California.

Spinach (Spinacia oleracea) plants exhibiting severe stunting and leaves that showed interveinal yellowing, thickening, and deformation were found in an experimental trial adjacent to an artichoke field in Monterey County, CA in October of 2008. Percent incidence of symptomatic plants ranged from 20 to 39% in cvs. Bordeaux, Lazio, and Tigercat. Symptomatic plants were positive for Impatiens necrotic spot virus (INSV; family Bunyaviridae, genus Tospovirus) and were negative for Tomato spotted wilt virus, Cucumber mosaic virus, and Tobacco mosaic virus when tested with immunostrips (Agdia Inc., Elkhart, IN). The INSV-positive spinach was used for mechanical transmission to Nicotiana benthamiana, Chenopodium quinoa, and spinach. All inoculated plants were positive for INSV with immunostrips. To further confirm the presence of INSV, reverse transcription (RT)-PCR was conducted. Total RNA was extracted from the symptomatic spinach plants using a RNeasy Plant Kit (Qiagen Inc., Valencia, CA) and used as a template in RT-PCR using forward (5'-GGATGTAAGCCCTTCTTTGTAGTGG-3') and reverse (5'-CCTTCCAAGTCACCCTCTGATTG-3') primers specific to the INSV nucleoprotein (N) gene (GenBank Accession No. DQ425096). Amplicons of the expected size (approximately 364 bp) were obtained from both field-infected and mechanically inoculated spinach plants. Four amplicons were sequenced and compared with INSV N gene sequence in GenBank to confirm the identity of the products. Sequences obtained had 99% nucleotide identity with INSV sequences available under the GenBank Accession Nos. L20885, DQ523597, DQ523598, X66872, L20886, D00914, AB109100, and DQ425096. INSV can be one of the most serious viral pathogens of ornamental plants in North America and Europe. The host range of INSV is expanding and recent reports of INSV infection of vegetables include lettuce, peppers, peanut, and potato (1-4). To our knowledge, this is the first report of natural occurrence of INSV in spinach in California. Since INSV is vectored by thrips, its expanding natural host could make it an economically important problem in California and the United States. References: (1) S. T. Koike et al. Plant Dis. 92:1248, 2008. (2) R. A. Naidu et al. Online publication. doi:10.1094/PHP-2005-0727-01-HN, Plant Health Progress, 2005. (3) S. S Pappu et al. Plant Dis. 83:966, 1999. (4) K. L. Perry et al. Plant Dis. 89:340, 2005.

High prevalence of albuminuria in population-based patients diagnosed with type 2 diabetes in the Shanghai downtown.

OBJECTIVE: The prevalence of albuminuria and the risk factors associated with albuminuria were evaluated among the Chinese patients diagnosed with type 2 diabetes aged over 30 in the Shanghai downtown. We also evaluated the variability of urinary albumin-to-creatinine ratio (ACR) among the three measurements and the relationship between diabetic retinopathy (DR) and albuminuria. METHODS: The 1039 Chinese patients diagnosed with type 2 diabetes aged over 30 were investigated by randomized cluster sampling in the Shanghai downtown and 1018 patients were analyzed in this study. Body mass measurements including height, weight, waist circumference and hip circumference, resting blood pressure, fasting blood measures, urinary ACR and the digitally stored fundus images were investigated. The prevalence of albuminuria was calculated and the risk factors associated with albuminuria were evaluated by stepwise logistic regression. The concordance of urinary ACR was evaluated by observed agreement. The relationship between albuminuria and DR was also evaluated. RESULTS: (1) The mean age of all patients was 66.10+/-11.54 years and the duration of diabetes was 7.89+/-7.16 years. (2) The prevalence of albuminuria was 49.6% among the Chinese patients diagnosed with type 2 diabetes aged over 30 in the Shanghai downtown, 41.4% with microalbuminuria and 8.2% with macroalbuminuria. (3) Microalbuminuria was significantly associated with systolic blood pressure, gender and waist circumference. Macroalbuminuria was significantly associated with systolic blood pressure and duration of diabetes. (4) Observed agreement among the three urinary ACR measurement for albuminuria staging was 73.3% (first versus second), 64.5% (first versus third) and 77.5% (second versus third). Observed agreement in the albuminuria staging between the single urinary ACR measurement and all three urinary ACR measurements was 85.8% (first versus all three), 87.6% (second versus all three) and 81.9% (third versus all three). (5) The percentage of DR in the macroalbuminuric group (59.2%) was significantly higher than that in the normalbuminuria group (16.1%) and microalbuminuria group (24.6%). (6) The macroalbuminuric patients with DR had significantly increased fasting blood glucose and HbA1c compared with the macroalbuminuric patients without DR. CONCLUSION: The prevalence of microalbuminuria observed in the Chinese patients diagnosed with type 2 diabetes aged over 30 in the Shanghai downtown reached up to 41.4% though the observations in our study might be representative of the diabetic patients of the Shanghai downtown. We agreed that at least two of the three urinary collections were done in a 3- to 6-month period because of the day-to-day variability in albumin excretion. The percentage of DR among the patients with macroalbuminuria was 59.2%, and the macroalbuminuric patients with the significantly high plasma glucose and DR were prone to diagnose DN.

Gene expression profiling in human insulinoma tissue: genes involved in the insulin secretion pathway and cloning of novel full-length cDNAs.

Insulinoma is a clinically common cause of organic hypoglycemia. The prominent characteristic of insulinoma is endogenous hyperinsulinism. Until now, the molecular biology of human insulinoma has been little understood. In this study, gene expression profiling of human insulinoma was established by expressed sequence tag (EST) sequencing and cDNA array. A total of 2063 clones were obtained, of these, 1589 clones were derived from EST sequencing, 975 clones were derived from cDNA array and 501 clones were shared by the two methods. G protein alpha-stimulating activity polypeptide (Gsalpha) and carboxypeptidase E (CPE) were the most highly expressed genes in human insulinoma, as derived by EST sequencing and cDNA array respectively. The genes involved in the protein/insulin secretion pathway were strongly expressed in human insulinoma tissue. Meanwhile, eight full-length cDNAs of novel genes were cloned and sequenced. The results demonstrated the molecular biology of human insulinoma tissue at the level of transcript abundance and validated the efficacy of EST sequencing combined with cDNA array in the construction of gene expression profiling. In conclusion, the predominance of the genes participating in the secretory pathway suggested that regulation of secretion might be a major mechanism by which insulin release is abnormally increased in patients with insulinomas. It was also concluded that overexpression of the Gsalpha gene played an important role in the pathogenesis of insulinoma.

[Application of modified shuttle like incision in benign skin tumour in the maxillofacial region].

OBJECTIVE: To design a modified shuttle-like incision for benign skin tumours in the maxillofacial region. METHODS: Some merits of the modified shuttle like incision was verified through experiment upon Cadavers and accurate mathematical calculation. Then it was used in the resection of 24 patients with benign skin tumour in the maxillofacial region. RESULTS: If the radius of the tumours were separately 0.5 cm, 1.0 cm and 1.5 cm, the modified shuttle-like incision can save the skin area by 17.22%,15.42% and 16.98%; no(or little) tension was found in sewing. The sutural lines were levelly concealed in the kraissl's lines and no"cat ear"was found after sewing. After 1-7 years of following up, all of the scars of the 24 cases were appraised as good. CONCLUSION: The modified shuttle-like incision was better than the traditional one. It can be performed widely in the clinical operation on benign skin tumours in the maxillofacial region.

A small asparagine-rich protein required for S-allele-specific pollen rejection in Nicotiana.

Although S-locus RNases (S-RNases) determine the specificity of pollen rejection in self-incompatible (SI) solanaceous plants, they alone are not sufficient to cause S-allele-specific pollen rejection. To identify non-S-RNase sequences that are required for pollen rejection, a Nicotiana alata cDNA library was screened by differential hybridization. One clone, designated HT, hybridized strongly to RNA from N. alata styles but not to RNA from Nicotiana plumbaginifolia, a species known to lack one or more factors necessary for S-allele-specific pollen rejection. Sequence analysis revealed a 101-residue ORF including a putative secretion signal and an asparagine-rich domain near the C terminus. RNA blot analysis showed that the HT-transcript accumulates in the stigma and style before anthesis. The timing of HT-expression lags slightly behind S(C10)-RNase in SI N. alata S(C10)S(C10) and is well correlated with the onset of S-allele-specific pollen rejection in the style. An antisense-HT construct was prepared to test for a role in pollen rejection. Transformed (N. plumbaginifolia x SI N. alata S(C10)S(C10)) hybrids with reduced levels of HT-protein continued to express S(C10)-RNase but failed to reject S(C10)-pollen. Control hybrids expressing both S(C10)-RNase and HT-protein showed a normal S-allele-specific pollen rejection response. We conclude that HT-protein is directly implicated in pollen rejection.

Exchanging sequence domains between S-RNases from Nicotiana alata disrupts pollen recognition.

In self-incompatible plants of the Solanaceae, the specificity of pollen rejection is controlled by a single multiallelic S-locus. Pollen tube growth is inhibited in the style when its single S-allele matches either S-allele present in the diploid pistil. Each S-allele encodes an S-RNase with a unique sequence. S-RNases are secreted into the extra-cellular matrix of the transmitting tract which guides pollen tubes toward the ovary. Although it is known that S-RNases are the determinants of S-allele specificity in the pistil, it is not known how allele-specific information is encoded in the sequence. Therefore, we exchanged domains between S-RNases with different recognition specificities and expressed the chimeric proteins in transgenic plants to determine their effects on pollination behavior. Nine chimeric constructs were prepared in which domains from Nicotiana alata SA2- and SC10-RNases were exchanged. Among these nine constructs, the entire S-RNase sequence was sampled by exchanging single variable domains as well as larger blocks of contiguous sequences. The chimeric S-RNases retained enzymatic activity and were expressed at levels comparable to control transformants expressing SA2- and SC10-RNases. However, none of the chimeric S-RNases caused rejection of either SA2- or SC10-pollen. We conclude that the recognition function of S-RNases can be disrupted by alterations in many parts of the sequence. It appears that the recognition function of S-RNase is not localized to a specific domain.

S RNase and Interspecific Pollen Rejection in the Genus Nicotiana: Multiple Pollen-Rejection Pathways Contribute to Unilateral Incompatibility between Self-Incompatible and Self-Compatible Species.

In self-incompatible (SI) plants, the S locus acts to prevent growth of self-pollen and thus promotes outcrossing within the species. Interspecific crosses between SI and self-compatible (SC) species often show unilateral incompatibility that follows the SI x SC rule: SI species reject pollen from SC species, but the reciprocal crosses are usually compatible. The general validity of the SI x SC rule suggests a link between SI and interspecific pollen rejection; however, this link has been questioned because of a number of exceptions to the rule. To clarify the role of the S locus in interspecific pollen rejection, we transformed several Nicotiana species and hybrids with genes encoding SA2 or SC10 RNase from SI N. alata. Compatibility phenotypes in the transgenic plants were tested using pollen from three SC species showing unilateral incompatibility with N. alata. S RNase was implicated in rejecting pollen from all three species. Rejection of N. plumbaginifolia pollen was similar to S allele-specific pollen rejection, showing a requirement for both S RNase and other genetic factors from N. alata. In contrast, S RNase-dependent rejection of N. glutinosa and N. tabacum pollen proceeded without these additional factors. N. alata also rejects pollen from the latter two species through an S RNase-independent mechanism. Our results implicate the S locus in all three systems, but it is clear that multiple mechanisms contribute to interspecific pollen rejection.

S-RNase expressed in transgenic Nicotiana causes S-allele-specific pollen rejection.

Many angiosperms employ self-incompatibility systems to prevent inbreeding. The simple genetics of such systems have made them attractive models of plant cellular communication. Implicit in the single locus genetics is that only one or a few gene products are necessary for recognition and rejection of incompatible pollen. Results in the sporophytic system of the Brassicaceae suggest that different S-locus products are responsible for the pollen and pistil parts of the recognition and rejection response. In solanaeceous plants, which have a gametophytic self-incompatibility system, the S locus product responsible for the pollen portion of the interaction has not been identified, but ribonucleases encoded by the S-locus (S-RNases) are strongly implicated in the style part of the recognition and rejection reaction. In Nicotiana alata, pollen recognition and rejection occur if its S-allele matches either S-allele in the style. The putative stylar S-RNase is abundant in the transmitting tract, and pollen rejection may be related to action of S-RNase on pollen RNAs. Efforts to understand the molecular basis for pollen recognition and rejection have been limited by the lack of a system for manipulating and expressing S-RNases. Here we use the promoter of a style-expressed gene from tomato to obtain high levels of S-RNase expression in transgenic Nicotiana. Recognition and rejection of N. alata pollen S-alleles occur faithfully in the transgenic plants. Our results show that S-RNases alone are sufficient for pollen rejection in this system.