Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Relative apparent synapomorphy analysis (RASA). I: The statistical measurement of phylogenetic signal

We have developed a new approach to the measurement of phylogenetic signal in character state matrices called relative apparent synapomorphy analysis (RASA). RASA provides a deterministic, statistical measure of natural cladistic hierarchy (phylogenetic signal) in character state matrices. The method works by determining whether a measure of the rate of increase of cladistic similarity among pairs of taxa as a function of phenetic similarity is greater than a null equiprobable rate of increase. Our investigation of the utility and limitations of RASA using simulated and bacteriophage T7 data sets indicates that the method has numerous advantages over existing measures of signal. A first advantage is computational efficiency. A second advantage is that RASA employs known methods of statistical inference, providing measurable sensitivity and power. The performance of RASA is examined under various conditions of branching evolution as the number of characters, character states per character, and mutations per branch length are varied. RASA appears to provide an unbiased and reliable measure of phylogenetic signal, and the general approach promises to be useful in the development of new techniques that should increase the rigor and reliability of phylogenetic estimates.      

Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis)

Analysis of the heat-shock proteins (hsps) of six closely related species of Poeciliopsis demonstrated the existence of biochemical diversity in the hsp100, hsp70, hsp60, and hsp30 protein families among species. Each species expressed five to seven hsp70-related isoforms. Constitutive 70-kD isoforms were identical among species, but four different patterns of heat-inducible isoforms were seen in these six species. Members of the hsp70 family of molecular chaperones are included among the most highly conserved proteins known, and the possibility of variation in hsp70 among closely related species has rarely been addressed. The hsp30 family is known to be less conserved than the hsp70 family, and, as expected, the Poeciliopsis hsp30 patterns showed more variation. Most of the hsp30 isoforms characteristic of a particular species were unique to that species. Hsp100 and hsp60 were identical in five of the species, but alternate isoforms were found in P. monacha. The small size and limited geographical distribution of the P. monacha population have probably contributed to the uniqueness of the monacha pattern. Two of the species were shown to acquire thermotolerance, the ability to withstand normally lethal temperatures when subjected to a gradual temperature increase. Rapid-heating protocols commonly used to establish critical thermal maxima of organisms do not include this inducible component of thermoresistance and therefore do not adequately assess an organism's capacity to withstand thermal stress.      

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

The Genetics of Leaf Flecking in Maize and Its Relationship to Plant Defense and Disease Resistance

Physiological leaf spotting, or flecking, is a mild-lesion phenotype observed on the leaves of several commonly used maize (Zea mays) inbred lines and has been anecdotally linked to enhanced broad-spectrum disease resistance. Flecking was assessed in the maize nested association mapping (NAM) population, comprising 4,998 recombinant inbred lines from 25 biparental families, and in an association population, comprising 279 diverse maize inbreds. Joint family linkage analysis was conducted with 7,386 markers in the NAM population. Genome-wide association tests were performed with 26.5 million single-nucleotide polymorphisms (SNPs) in the NAM population and with 246,497 SNPs in the association population, resulting in the identification of 18 and three loci associated with variation in flecking, respectively. Many of the candidate genes colocalizing with associated SNPs are similar to genes that function in plant defense response via cell wall modification, salicylic acid- and jasmonic acid-dependent pathways, redox homeostasis, stress response, and vesicle trafficking/remodeling. Significant positive correlations were found between increased flecking, stronger defense response, increased disease resistance, and increased pest resistance. A nonlinear relationship with total kernel weight also was observed whereby lines with relatively high levels of flecking had, on average, lower total kernel weight. We present evidence suggesting that mild flecking could be used as a selection criterion for breeding programs trying to incorporate broad-spectrum disease resistance.The plant hypersensitive response (HR) is a form of programmed cell death (PCD) characterized by rapid, localized cell death at the point of attempted pathogen penetration, usually resulting in disease resistance (Coll et al., 2011). It is often associated with other responses, including ion fluxes, an oxidative burst, lipid peroxidation, and cell wall fortification (Hammond-Kosack and Jones, 1996). van Doorn et al. (2011) suggested that HR is a type of PCD sharing features with, but distinct from, both vacuolar cell death and necrosis.HR has been associated with resistance to almost every class of pathogen and pest, including bacteria, viruses, fungi, nematodes, insects, and parasitic plants (Wu and Baldwin, 2010), and generally is most effective against biotrophic pathogens, since biotrophs require a long-term feeding relationship with living host cells. It is generally mediated by dominant resistance (R) genes whose activation is triggered by the direct or indirect detection of specific pathogen-derived effector proteins (Bent and Mackey, 2007). R proteins are maintained in their inactive state if their corresponding effector is not present. Mutants in which HR is constitutively active have been identified in many plant species, including maize/corn (Zea mays; Walbot et al., 1983; Johal, 2007), Arabidopsis (Arabidopsis thaliana; Lorrain et al., 2003), barley (Hordeum vulgare; Wolter et al., 1993), and rice (Oryza sativa; Yin et al., 2000).One well-known class of plant mutants spontaneously form lesions (patches of dead or chlorotic cells) in the absence of any obvious injury, stress, or infection to the plant. Since these lesions in some cases resemble HR, they have been termed disease-lesion mimics (Neuffer and Calvert, 1975). These mutants, which we will here collectively term Les mutants, have been studied extensively, especially in maize (Walbot et al., 1983; Johal et al., 1995; Johal, 2007) and Arabidopsis (Coll et al., 2011). While some of these lesion phenotypes are indeed caused by perturbations in the plant defense response (Hu et al., 1996; Rustérucci et al., 2001), some of the genes underlying this mutant class affect various other pathways that cause cell death if their function is perturbed (Johal, 2007). For instance, the Arabidopsis gene acd2 and the maize gene lls1 are defective in chlorophyll degradation (Gray et al., 1997; Mach et al., 2001).We have defined leaf flecking as the mild, genetically determined spotting observed on many maize inbred cultivars (Vontimitta et al., 2015; Fig. 1). The trait is qualitatively and visually similar to, but quantitatively less severe than, Les mutant phenotypes. The distinction between what constitutes a flecking versus a mild Les trait is necessarily somewhat arbitrary, but for our purposes, we have defined any nonproliferating and distinct leaf-spotting phenotype as flecking.Open in a separate windowFigure 1.A, Examples of variation in the flecking phenotype among inbred lines, with severity increasing from left to right (flecking scores in parentheses, from 0 to 4, scored on a scale of 1–10). B, Leaves of the lines nearly isogenic to inbred Mo20W, into which specific indicated dominant Les mutant genes have been introgressed (Rp1-D21 mutation in an H95 inbred background). Photographs were taken in Clayton, North Carolina, 12 weeks after planting. This figure is adapted from Figure 1 of Vontimitta et al. (2015).Leaf flecking is familiar to most corn breeders, appearing in such well-known and widely used lines such as Mo17 (Zehr et al., 1994) and in several other species such as barley (Makepeace et al., 2007), wheat (Triticum aestivum; Nair and Tomar, 2001), and oat (Avena sativa; Ferdinandsen and Winge, 1930). Flecking tends to be more noticeable in inbreds compared with their derived hybrids (M. Goodman and W. Dolezal, personal communication). Anecdotally, it is often thought to be indicative of a constitutive low-level defense response and as a marker for increased disease resistance.In previous work, we and others have defined the genetic architectures associated with resistance to several maize diseases, including southern leaf blight (SLB; causal agent, Cochliobolus heterostrophus), northern leaf blight (NLB; causal agent, Exserohilum turcicum), and gray leaf spot (GLS; causal agent, Cercospora zeae-maydis; Kump et al., 2011; Poland et al., 2011; Wisser et al., 2011; Benson et al., 2015), and with the control of the maize HR (Chintamanani et al., 2010; Chaikam et al., 2011; Olukolu et al., 2013). For much of this work, we used two powerful mapping populations: the maize association population (Flint-Garcia et al., 2005), a collection of 302 diverse inbred lines with low linkage disequilibrium, and the 5,000-line nested association mapping (NAM) population (McMullen et al., 2009), which is made up of 25 200-line recombinant inbred line (RIL) subpopulations derived from crosses between the common parent B73 and 25 diverse inbreds. Using these populations, it is possible to both sample a diverse array of germplasm and map quantitative trait loci (QTLs) precisely, in some cases to the gene level (Tian et al., 2011; Cook et al., 2012; Hung et al., 2012; Larsson et al., 2013; Olukolu et al., 2013; Wang and Balint-Kurti, 2016).A recent study using 300 lines from the maize intermated B73 × Mo17 population advanced intercross line mapping population identified low but moderately significant positive correlations between increased flecking and increased disease resistance and defense response (Vontimitta et al., 2015). Loci associated with variation in flecking were mapped, although these loci did not colocalize with QTLs identified previously for disease resistance and defense response traits (Balint-Kurti et al., 2007, 2008, 2010; Olukolu et al., 2013). In this study, we have extended this work to examine the genetic basis of leaf flecking over a much more diverse set of maize germplasm using a substantially larger population. We mapped loci associated with variation in leaf flecking and identified candidate genes and pathways that may be involved in this phenotype. Additionally, we have examined the correlations between leaf flecking and disease resistance, the hypersensitive defense response, and total kernel weight.