QTL and PACE analyses identify candidate genes for anthracnose resistance in tomato.

Anthracnose, caused by the fungal pathogen Colletotrichum spp., is one of the most significant tomato diseases in the United States and worldwide. No commercial cultivars with anthracnose resistance are available, limiting resistant breeding. Cultivars with genetic resistance would significantly reduce crop losses, reduce the use of fungicides, and lessen the risks associated with chemical application. A recombinant inbred line (RIL) mapping population (N=243) has been made from a cross between the susceptible US28 cultivar and the resistant but semiwild and small-fruited 95L368 to identify quantitative trait loci (QTLs) associated with anthracnose resistance. The RIL population was phenotyped for resistance by inoculating ripe field-harvested tomato fruits with Colletotrichum coccodes for two seasons. In this study, we identified twenty QTLs underlying resistance, with a range of phenotypic variance of 4.5 to 17.2% using a skeletal linkage map and a GWAS. In addition, a QTLseq analysis was performed using deep sequencing of extreme bulks that validated QTL positions identified using traditional mapping and resolved candidate genes underlying various QTLs. We further validated AP2-like ethylene-responsive transcription factor, N-alpha-acetyltransferase (NatA), cytochrome P450, amidase family protein, tetratricopeptide repeat, bHLH transcription factor, and disease resistance protein RGA2-like using PCR allelic competitive extension (PACE) genotyping. PACE assays developed in this study will enable high-throughput screening for use in anthracnose resistance breeding in tomato.

Steroidal glycoalkaloids contribute to anthracnose resistance in Solanum lycopersicum.

Anthracnose is a widespread plant disease caused by various species of the fungal pathogen Colletotrichum. In solanaceous plants such as tomato (Solanum lycopersicum), Colletotrichum infections exhibit a quiescent, asymptomatic state in developing fruit, followed by a transition to necrotrophic infections in ripe fruit. Through analysis of fruit tissue extracts of 95L368, a tomato breeding line that yields fruit with enhanced anthracnose resistance, we identified a role for steroidal glycoalkaloids (SGAs) in anthracnose resistance. The SGA α-tomatine and several of its derivatives accumulated at higher levels, in comparison with fruit of the susceptible tomato cultivar US28, and 95L368 fruit extracts displayed fungistatic activity against Colletotrichum. Correspondingly, ripe and unripe 95L368 fruit displayed enhanced expression of glycoalkaloid metabolic enzyme (GAME) genes, which encode key enzymes in SGA biosynthesis. Metabolomics analysis incorporating recombinant inbred lines generated from 95L368 and US28 yielded strong positive correlations between anthracnose resistance and accumulation of α-tomatine and several derivatives. Lastly, transient silencing of expression of the GAME genes GAME31 and GAME5 in anthracnose-susceptible tomato fruit yielded enhancements to anthracnose resistance. Together, our data support a role for SGAs in anthracnose defense in tomato, with a distinct SGA metabolomic profile conferring resistance to virulent Colletotrichum infections in ripe fruit.

The flowering time regulator FLK controls pathogen defense in Arabidopsis thaliana.

Plant disease resistance is a complex process that is maintained in an intricate balance with development. Increasing evidence indicates the importance of posttranscriptional regulation of plant defense by RNA binding proteins. In a genetic screen for suppressors of Arabidopsis (Arabidopsis thaliana) accelerated cell death 6-1 (acd6-1), a small constitutive defense mutant whose defense level is grossly in a reverse proportion to plant size, we identified an allele of the canonical flowering regulatory gene FLOWERING LOCUS K HOMOLOGY DOMAIN (FLK) encoding a putative protein with triple K homology (KH) repeats. The KH repeat is an ancient RNA binding motif found in proteins from diverse organisms. The relevance of KH-domain proteins in pathogen resistance is largely unexplored. In addition to late flowering, the flk mutants exhibited decreased resistance to the bacterial pathogen Pseudomonas syringae and increased resistance to the necrotrophic fungal pathogen Botrytis cinerea. We further found that the flk mutations compromised basal defense and defense signaling mediated by salicylic acid (SA). Mutant analysis revealed complex genetic interactions between FLK and several major SA pathway genes. RNA-seq data showed that FLK regulates expression abundance of some major defense- and development-related genes as well as alternative splicing of a number of genes. Among the genes affected by FLK is ACD6, whose transcripts had increased intron retentions influenced by the flk mutations. Thus, this study provides mechanistic support for flk suppression of acd6-1 and establishes that FLK is a multifunctional gene involved in regulating pathogen defense and development of plants.

Hemodynamic actions of acute ethanol after resuscitation from traumatic brain injury.

BACKGROUND: The purposes of this study were to determine how clinically relevant levels of acute ethanol (EtOH) influence cerebral perfusion pressure (CPP), cerebral venous O saturation (Scvo ), and systemic hemodynamics after fluid resuscitation from traumatic brain injury (TBI); and to test the hypothesis that the actions of EtOH on these variables are mediated by adenosine. METHODS: Anesthetized swine were ventilated (Fio = 0.4) and instrumented. In protocol 1, EtOH (3.5 g/kg, n = 11) or its vehicle (n = 17) was administered orally before TBI + 40% hemorrhage. At 90 minutes post-TBI, resuscitation consisted of shed blood + saline. In protocol 2, either saline (n = 15) or an adenosine-regulating agent (5-amino-4-imidazolecarboxamide riboside) in saline (1 mg/kg bolus + 12 mg/kg/h intravenously [i.v.]) (n = 5), was administered i.v. before TBI + 45% hemorrhage. At 90 minutes post-TBI, resuscitation consisted of saline only (three times shed blood volume). In protocol 3, EtOH was administered i.v. (1 g/kg; 20% vol/vol in saline) followed by either an adenosine receptor antagonist (theophylline, 10 mg/kg) or an adenosine uptake inhibitor (dipyridamole, 0.25 mg/kg). RESULTS: In protocol 1, with no EtOH, 11 of 17 (65%) survived post-TBI hypotension. Mean arterial blood pressure, cardiac index, and mixed venous oxygen saturation were stable for 1 hour at 40% to 60% below their respective baselines, whereas lactate increased three- to fourfold (all p < 0.05). After fluid resuscitation, most variables rapidly corrected, but intracranial pressure was increased 10 to 15 mm Hg (p < 0.05). With EtOH, 9 of 11 (82%) survived post-TBI hypotension (p = 0.42 vs. no EtOH). After resuscitation from TBI, there were significant effects of EtOH on systemic hemodynamics (mean arterial pressure, cardiac index, mixed venous oxygen saturation), on CPP, on lactate, and on Scvo at normo- and hypercapnia (all p < 0.05). The data from protocol 2 showed that essentially none of these changes were duplicated with an adenosine-regulating agent. In protocol 3, i.v EtOH produced small but significant changes in Scvo, intracranial pressure, and lactate, at normo-, hyper-, and hypocapnia. Dipyridamole and theophylline tended to have opposite, albeit small and not statistically significant, effects on these variables relative to EtOH alone.(2) (2) CONCLUSION: Acute EtOH (200-300 mg/dL) did not increase mortality after TBI + secondary hypotension, as long as cardiopulmonary support was provided. With EtOH, CPP was maintained and cerebral blood flow appeared to be adequate, if not excessive, with respect to cerebral metabolic demand, as judged by changes in Scvo at normo-, hyper-, and hypocapnia. These changes were probably not mediated, but might have been modulated, by increases in endogenous adenosine.