Effects of virus infection on pollen production and pollen performance: Implications for the spread of resistance alleles.

PREMISE OF STUDY: Studies over the past 25 years have shown that environmental stresses adversely affect male function, including pollen production and pollen performance (germination and pollen tube growth rate). Consequently, genetic variation among plants in resistance to a stress has the potential to impact pollen donation to conspecifics and, if deposited onto a stigma, the ability of the pollen to achieve fertilization. We examined the effects of a nonlethal virus epidemic on pollen production and pollen performance in a population of susceptible and resistant (transgenic) wild squash (Cucurbita pepo subsp. texana). METHODS: We grew 135 susceptible and 45 virus-resistant wild squash plants in each of two 0.4-ha fields, initiated a zucchini yellow mosaic virus (ZYMV) epidemic, and recorded staminate and pistillate flower production per plant over the field season and the total number of mature fruit. We also assessed pollen production per flower on ZYMV-infected and non-infected plants and the ability of pollen from flowers on infected and non-infected plants to achieve fertilization under competitive conditions. KEY RESULTS: ZYMV infection reduced flower and fruit production per plant and pollen production per flower. Pollen from infected plants was also less likely to sire a seed under competitive conditions. CONCLUSIONS: ZYMV infection adversely impacts the amount of pollen that can be donated to conspecifics, and pollen competition within the styles increases the probability that the ovules are fertilized by pollen from plants that are thriving when challenged by a viral disease.

Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus.

When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL (SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NAD(+)) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD(+), NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.

Structure and protective efficacy of the Staphylococcus aureus autocleaving protease EpiP.

Despite the global medical needs associated with Staphylococcus aureus infections, no licensed vaccines are currently available. We identified and characterized a protein annotated as an epidermin leader peptide processing serine protease (EpiP), as a novel S. aureus vaccine candidate. In addition, we determined the structure of the recombinant protein (rEpiP) by X-ray crystallography. The crystal structure revealed that rEpiP was cleaved somewhere between residues 95 and 100, and we found that the cleavage occurs through an autocatalytic intramolecular mechanism. The protein expressed by S. aureus cells also appeared to undergo a similar processing event. To determine whether the protein acts as a serine protease, we mutated the hypothesized catalytic serine 393 residue to alanine, generating rEpiP-S393A. The crystal structure of this mutant protein showed that the polypeptide chain was not cleaved and was not interacting stably with the active site. Indeed, rEpiP-S393A was shown to be impaired in its protease activity. Mice vaccinated with rEpiP were protected from S. aureus infection (34% survival, P=0.0054). Moreover, the protective efficacy generated by rEpiP and rEpiP-S393A was comparable, implying that the noncleaving mutant could be used for vaccination purposes.

Large scale structural rearrangement of a serine hydrolase from Francisella tularensis facilitates catalysis.

Tularemia is a deadly, febrile disease caused by infection by the gram-negative bacterium, Francisella tularensis. Members of the ubiquitous serine hydrolase protein family are among current targets to treat diverse bacterial infections. Herein we present a structural and functional study of a novel bacterial carboxylesterase (FTT258) from F. tularensis, a homologue of human acyl protein thioesterase (hAPT1). The structure of FTT258 has been determined in multiple forms, and unexpectedly large conformational changes of a peripheral flexible loop occur in the presence of a mechanistic cyclobutanone ligand. The concomitant changes in this hydrophobic loop and the newly exposed hydrophobic substrate binding pocket suggest that the observed structural changes are essential to the biological function and catalytic activity of FTT258. Using diverse substrate libraries, site-directed mutagenesis, and liposome binding assays, we determined the importance of these structural changes to the catalytic activity and membrane binding activity of FTT258. Residues within the newly exposed hydrophobic binding pocket and within the peripheral flexible loop proved essential to the hydrolytic activity of FTT258, indicating that structural rearrangement is required for catalytic activity. Both FTT258 and hAPT1 also showed significant association with liposomes designed to mimic bacterial or human membranes, respectively, even though similar structural rearrangements for hAPT1 have not been reported. The necessity for acyl protein thioesterases to have maximal catalytic activity near the membrane surface suggests that these conformational changes in the protein may dually regulate catalytic activity and membrane association in bacterial and human homologues.

Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria.

Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpS(SA)), Vibrio cholerae (AcpS(VC)) and Bacillus anthracis (AcpS(BA)) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpS(BA) is emphasized because of the two 3',5'-adenosine diphosphate (3',5'-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3',5'-ADP is bound as the 3',5'-ADP part of CoA in the known structures of the CoA-AcpS and 3',5'-ADP-AcpS binary complexes. The position of the second 3',5'-ADP has never been described before. It is in close proximity to the first 3',5'-ADP and the ACP-binding site. The coordination of two ADPs in AcpS(BA) may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.

Indirect costs of a nontarget pathogen mitigate the direct benefits of a virus-resistant transgene in wild Cucurbita.

Virus-resistant transgenic squash are grown throughout the United States and much of Mexico and it is likely that the virus-resistant transgene (VRT) has been introduced to wild populations repeatedly. The evolutionary fate of any resistance gene in wild populations and its environmental impacts depend upon trade-offs between the costs and benefits of the resistance gene. In a 3-year field study using a wild gourd and transgenic and nontransgenic introgressives, we measured the effects of the transgene on fitness, on herbivory by cucumber beetles, on the incidence of mosaic viruses, and on the incidence of bacterial wilt disease (a fatal disease vectored by cucumber beetles). In each year, the first incidence of zucchini yellow mosaic virus occurred in mid-July and spread rapidly through the susceptible plants. We found that the transgenic plants had greater reproduction through both male and female function than the susceptible plants, indicating that the VRT has a direct fitness benefit for wild gourds under the conditions of our study. Moreover, the VRT had no effect on resistance to cucumber beetles or the incidence of wilt disease before the spread of the virus. However, as the virus spread through the fields, the cucumber beetles became increasingly concentrated upon the healthy (mostly transgenic) plants, which increased exposure to and the incidence of wilt disease on the transgenic plants. This indirect cost of the VRT (mediated by a nontarget herbivore and pathogen) mitigated the overall beneficial effect of the VRT on fitness.

GTP hydrolysis promotes disassembly of the atlastin crossover dimer during ER fusion.

Membrane fusion of the ER is catalyzed when atlastin GTPases anchored in opposing membranes dimerize and undergo a crossed over conformational rearrangement that draws the bilayers together. Previous studies have suggested that GTP hydrolysis triggers crossover dimerization, thus directly driving fusion. In this study, we make the surprising observations that WT atlastin undergoes crossover dimerization before hydrolyzing GTP and that nucleotide hydrolysis and Pi release coincide more closely with dimer disassembly. These findings suggest that GTP binding, rather than its hydrolysis, triggers crossover dimerization for fusion. In support, a new hydrolysis-deficient atlastin variant undergoes rapid GTP-dependent crossover dimerization and catalyzes fusion at an initial rate similar to WT atlastin. However, the variant cannot sustain fusion activity over time, implying a defect in subunit recycling. We suggest that GTP binding induces an atlastin conformational change that favors crossover dimerization for fusion and that the input of energy from nucleotide hydrolysis promotes complex disassembly for subunit recycling.

The crossover conformational shift of the GTPase atlastin provides the energy driving ER fusion.

The homotypic fusion of endoplasmic reticulum membranes is catalyzed by the atlastin GTPase. The mechanism involves trans-dimerization between GTPase heads and a favorable crossover conformational shift, catalyzed by GTP hydrolysis, that converts the dimer from a "prefusion" to "postfusion" state. However, whether crossover formation actually energizes fusion remains unclear, as do the sequence of events surrounding it. Here, we made mutations in atlastin to selectively destabilize the crossover conformation and used fluorescence-based kinetic assays to analyze the variants. All variants underwent dimerization and crossover concurrently, and at wild-type rates. However, certain variants were unstable once in the crossover dimer conformation, and crossover dimer stability closely paralleled lipid-mixing activity. Tethering, however, appeared to be unimpaired in all mutant variants. The results suggest that tethering and lipid mixing are catalyzed concurrently by GTP hydrolysis but that the energy requirement for lipid mixing exceeds that for tethering, and the full energy released through crossover formation is necessary for fusion.

Multigenerational effects of inbreeding in Cucurbita pepo ssp. texana (Cucurbitaceae).

The shape of the fitness function relating the decline in fitness with coefficient of inbreeding (f) can provide evidence concerning the genetic basis of inbreeding depression, but few studies have examined inbreeding depression across a range of f using noncultivated species. Futhermore, studies have rarely examined the effects of inbreeding depression in the maternal parent on offspring fitness. To estimate the shape of the fitness function, we examined the relationship between f and fitness across a range off from 0.000 to 0.875 for components of both male and female fitness in Cucurbita pepo ssp. texana. Each measure of female fitness declined with f, including pistillate flower number, fruit number, seed number per fruit, seed mass per fruit, and percentage seed germination. Several aspects of male fitness also declined with f, including staminate flower number, pollen number per flower, and the number of days of flowering, although cumulative inbreeding depression was less severe for male (0.34) than for female function (0.39). Fitness tended to decline linearly with f between f = 0.00 and f = 0.75 for most traits and across cumulative lifetime fitness (mean = 0.66), suggesting that individual genes causing inbreeding depression are additive and the result of many alleles of small effect. However, most traits also showed a small reduction in inbreeding depression between f = 0.75 and f = 0.875, and evidence of purging or diminishing epistasis was found for in vitro pollen-tube growth rate. To examine inbreeding depression as a maternal effect, we performed outcross pollinations on f = 0.0 and f = 0.5 mothers and found that depression due to maternal inbreeding was 0.07, compared to 0.10 for offspring produced through one generation of selfing. In at least some families, maternal inbreeding reduced fruit number, seed number and mass, staminate flower number, pollen diameter, and pollen-tube growth rate. Collectively these results suggest that, while the fitness function appears to be largely linear for most traits, maternal effects may compound the effects of inbreeding depression in multigenerational studies, though this may be partially offset by purging or diminishing epistasis.

Resistance and tolerance to herbivory changes with inbreeding and ontogeny in a wild gourd (Cucurbitaceae).

Herbivory is a ubiquitous component of terrestrial communities that reduces plant growth and reproduction. Consequently, a goal of evolutionary ecology is to identify the causes and consequences of variation in herbivory within plant populations. This three-year study examined the effects of inbreeding on the resistance of wild gourd plants (Cucurbita pepo subsp. texana) to herbivory by cucumber beetles and the impact of the timing of herbivory on reproduction. We grew families of inbred and outbred gourds and recorded beetle damage at three developmental stages, incidence of beetle-vectored wilt disease, survival, and reproduction. While total beetle damage significantly depressed flower and fruit production, damage until mid-July did not depress any measure of reproduction, indicating that these gourds are tolerant of moderate levels of herbivory for most of the growing season. However, beetle damage accumulating after mid-July significantly depressed reproduction, indicating that plants have reduced tolerance during peak reproduction. Early damage, however, did increase the probability of contracting a deadly wilt disease that is vectored by the beetles, suggesting that tolerance and resistance are not alternative defense strategies. Inbreeding significantly reduced resistance to herbivory and, independently of beetle damage, reproductive output. Finally, we found additive genetic variation for both resistance and tolerance that varies with ontogeny.

A comparison of male and female responses to inbreeding in Cucurbita pepo subsp. texana (Cucurbitaceae).

Accurate estimates of inbreeding depression are necessary in order to predict the evolutionary dynamics of a population, but many studies estimate inbreeding depression based solely on components of female function such as fruit set, seed set, and seed quality. Because total fitness is achieved through both male and female functions in hermaphroditic plants, estimates of both male and female fitness are needed to estimate accurately the magnitude of inbreeding depression. Seedlings of a wild gourd, Cucurbita pepo subsp. texana, with coefficients of inbreeding of 0 and 0.75 were planted in an experimental garden, and several components of male and female fitness were measured over the course of the growing season. Fitness in inbred plants was confounded by both maternal and genetic inbreeding effects. Inbred individuals produced significantly fewer fruits than outcrossed individuals, and percentage germination of seeds from inbred individuals was significantly lower than seeds from outcrossed individuals. Inbred plants also produced significantly fewer staminate flowers and marginally fewer and smaller pollen grains per flower. Pollen from inbred plants also grew significantly more slowly in vitro than pollen from outcrossed plants. Multiplicative estimates of inbreeding depression revealed inbreeding depression for both male and female functions in wild gourd, but inbreeding depression through female function was stronger than inbreeding depression through male function.

Environmental variation influences the magnitude of inbreeding depression in Cucurbita pepo ssp. texana (Cucurbitaceae).

We grew inbred and outcrossed Cucurbita pepo ssp. texana plants and measured inbreeding depression for several male and female fitness traits 4 years in a row in adjacent fields at the same field station under the same cultivation conditions. We found that the magnitude of inbreeding depression varied from 0.16 to 0.53 from year to year and that those traits which were most affected tended to vary with year. We also grew inbred and outcrossed C. pepo ssp. texana plants in two adjacent fields differing only in the presence of nitrogen fertilizer to examine the effect of nutrient limitation as a form of environmental stress on the magnitude of inbreeding depression. We found that inbreeding depression was more severe in the unfertilized field. Overall, this study illustrates the notion that any estimate of inbreeding depression represents a single point in a cluster of possible estimates that can vary (often dramatically) with growing conditions.

LOTUS CORNICULATUS REGULATES OFFSPRING QUALITY THROUGH SELECTIVE FRUIT ABORTION.

Each inflorescence on Lotus corniculatus commonly aborts about half of its immature fruits. Compared to random patterns of fruit abortion, natural patterns of fruit abortion produce mature fruits that contain significantly more seeds. Moreover, these progeny are more likely to germinate, are more vigorous as seedlings, and have greater reproductive output as adults. These results indicate that L. corniculatus selectively aborts those fruits with the fewest seeds and, by doing so, increases the average quality of its offspring.

Inbreeding influences herbivory in Cucurbita pepo ssp. texana (Cucurbitaceae).

In a series of field experiments Diabrotica beetle herbivory was found to influence the magnitude of inbreeding depression in Cucurbita pepo ssp. texana, an annual monoecious vine. Beetles damage flowers and fruits and chew dime-sized holes in leaf tissue between major veins. Inbred plants were found to be more likely to be damaged by beetles and to have more leaves damaged per plant than outcrossed plants. A positive linear association was found between the coefficient of inbreeding and the magnitude of leaf damage, whereas a negative association was found between coefficient of inbreeding and several male and female fitness traits. When pesticides were used to control beetle herbivory, the interaction between coefficient of inbreeding and pesticide treatment was significant for fruit production and marginally significant for pollen quantity per anther. Therefore, the magnitude of inbreeding depression in C. pepo ssp. texana varies depending on the severity of beetle herbivory.

Pollen performance before and during the autotrophic-heterotrophic transition of pollen tube growth.

For species with bicellular pollen, the attrition of pollen tubes is often greatest where the style narrows at the transition between stigmatic tissue and the transmitting tissue of the style. In this region, the tubes switch from predominantly autotrophic to predominantly heterotrophic growth, the generative cell divides, the first callose plugs are produced, and, in species with RNase-type self-incompatibility (SI), incompatible tubes are arrested. We review the literature and present new findings concerning the genetic, environmental and stylar influences on the performance of pollen before and during the autotrophic-heterotrophic transition of pollen tube growth. We found that the ability of the paternal sporophyte to provision its pollen during development significantly influences pollen performance during the autotrophic growth phase. Consequently, under conditions of pollen competition, pollen selection during the autotrophic phase is acting on the phenotype of the paternal sporophyte. In a field experiment, using Cucurbita pepo, we found broad-sense heritable variation for herbivore-pathogen resistance, and that the most resistant families produced larger and better performing pollen when the paternal sporophytes were not protected by insecticides, indicating that selection during the autotrophic phase can act on traits that are not expressed by the microgametophyte. In a study of a weedy SI species, Solanum carolinense, we found that the ability of the styles to arrest self-pollen tubes at the autotrophic-heterotrophic transition changes with floral age and the presence of developing fruits. These findings have important implications for selection at the level of the microgametophyte and the evolution of mating systems of plants.

POLLEN LOADS AND PROGENY VIGOR IN CUCURBITA PEPO: THE NEXT GENERATION.

Previous research on the Black Beauty bush cv. of zucchini has documented a strong positive relationship between the size of the pollen load and the vigor (performance) of the progeny. Here we report the results of three studies designed to test the hypothesis that the previously observed differences in progeny vigor are heritable. Two studies examined the transmission of the pollen load effect to subsequent generations through the ovules (female role). The third study determined if there is genetic variation for pollen performance and if the pollen load effect could be transmitted to a subsequent generation through the pollen (male role). In each of these studies the vigor of the progeny from the subsequent generation was evaluated in the greenhouse and/or the field. The results of these studies reveal (1) that the ability to sire seeds does respond to selection imposed by high pollen loads, (2) that only 23 of the 35 total traits that we measured in the three studies of transmission to subsequent generations changed in the direction predicted by the pollen competition hypothesis, (3) that only 5 of the 35 traits were significantly affected by the size of the pollen load that produced the previous generation (but all 5 were in the direction predicted by the pollen competition hypothesis), and (4) that only one study produced an overall significant difference (MANOVA) attributable to the size of the pollen load that produced the previous generation (but it too was in the direction predicted by the pollen competition hypothesis). From these experiments we conclude that pollen competition appears to play a real but minor role in the production of differences in vigor between progeny arising from low versus high pollen loads. In Black Beauty bush cv. of zucchini, maternal effects, pollen-pistil interactions, or nonrandom patterns of seed abortion must play important roles as well.