It is widely accepted that spermatozoa are translationally silent. The present study demonstrates, for the first time, incorporation of labeled amino acids into polypeptides during sperm capacitation, which was completely inhibited by mitochondrial translation inhibitors but not by the cytoplasmic translation inhibitor. Unlike 80S cytoplasmic ribosomes, 55S mitochondrial ribosomes were present in polysomal fractions, indicating that these ribosomes are actively involved in protein translation in spermatozoa. Inhibition of protein translation significantly reduced sperm motility, capacitation and in vitro fertilization rate. Thus, contrary to the accepted dogma, nuclear genes are expressed as proteins in sperm during their residence in the female reproductive tract until fertilization.Supplemental material is available at http://genesdev.org.

In search of cell death-inducing proteins, we found a xyloglucanase (BcXYG1) that induced strong necrosis and a resistance response in dicot plants. Expression of the gene was strongly induced during the first 12 h post inoculation, and analysis of disease dynamics using PathTrack showed that a strain overexpressing produced early local necrosis, supporting a role of BcXYG1 as an early cell death-inducing factor. The xyloglucanase activity of BcXYG1 was not necessary for the induction of necrosis and plant resistance, as a mutant of BcXYG1 lacking the xyloglucanase enzymatic activity retained both functions. Residues in two exposed loops on the surface of BcXYG1 were found to be necessary for the induction of cell death but not to induce plant resistance. Further analyses showed that BcXYG1 is apoplastic and possibly interacts with the proteins of the plant cell membrane and also that the BcXYG1 cell death-promoting signal is mediated by the leucine-rich repeat receptor-like kinases BAK1 and SOBIR1. Our findings support the role of cell death-inducing proteins in establishing the infection of necrotrophic pathogens and highlight the recognition of fungal apoplastic proteins by the plant immune system as an important mechanism of resistance against this class of pathogens.

Necrotrophic fungi are unable to occupy living plant cells. How such pathogens survive first contact with living host tissue and initiate infection is therefore unclear. Here, we show that the necrotrophic grey mold fungus Botrytis cinerea undergoes massive apoptotic-like programmed cell death (PCD) following germination on the host plant. Manipulation of an anti-apoptotic gene BcBIR1 modified fungal response to PCD-inducing conditions. As a consequence, strains with reduced sensitivity to PCD were hyper virulent, while strains in which PCD was over-stimulated showed reduced pathogenicity. Similarly, reduced levels of PCD in the fungus were recorded following infection of Arabidopsis mutants that show enhanced susceptibility to B. cinerea. When considered together, these results suggest that Botrytis PCD machinery is targeted by plant defense molecules, and that the fungal anti-apoptotic machinery is essential for overcoming this host-induced PCD and hence, for establishment of infection. As such, fungal PCD machinery represents a novel target for fungicides and antifungal drugs.

Mammalian spermatozoa should reside in the female reproductive tract for a certain time before gaining the ability to fertilize. During this time, the spermatozoa undergo a series of biochemical processes collectively called capacitation. We recently demonstrated that actin polymerization is a necessary step in the cascade leading to capacitation. We demonstrate here for the first time a role for phospholipase D (PLD) in the induction of actin polymerization and capacitation in spermatozoa. The involvement of PLD is supported by specific inhibition of F-actin formation during sperm capacitation by PLD inhibitors and the stimulation of fast F-actin formation by exogenous PLD or phosphatidic acid (PA). Moreover, PLD activity is enhanced during capacitation before actin polymerization. Protein kinase A (PKA), known to be active in sperm capacitation, and protein kinase C (PKC), involved in the acrosome reaction, can both activate PLD and actin polymerization. We suggest that PKA- and PKC-dependent signal transduction pathways can potentially lead to PLD activation; however, under physiological conditions, actin polymerization depends primarily on PKA activity. Activation of PKA during capacitation causes inactivation of phospholipase C, and as a result, PKC activation is prevented. It appears that PKA activation promotes sperm capacitation whereas early activation of PKC during capacitation would jeopardize this process.

No abstract

Endophytes have profound impacts on plants, including beneficial effects on agriculturally important traits. We hypothesized that endophytes in wild plants include beneficial endophytes that are absent or underrepresented in domesticated crops. In this work, we studied the structure of endophyte communities in wheat-related grasses, Triticum dicoccoides and Aegilops sharonensis, and compared it to an endophyte community from wheat (T. aeastivum). Endophytes were isolated by cultivation and by cultivation-independent methods. In total, 514 intergenic spacer region sequences from single cultures were analyzed. Categorization at 97% sequence similarity resulted in 67 operational taxonomic units (OTUs) that were evenly distributed between the different plant species. A narrow core community of Alternaria spp. was found in all samples, but each plant species also contained a significant portion of unique endophytes. The cultivation-independent analysis identified a larger number of OTUs than the cultivation method, half of which were singletons or doubletons. For OTUs with a relative abundance >0.5%, similar numbers were obtained by both methods. Collectively, our data show that wild grass relatives of wheat contain a wealth of taxonomically diverse fungal endophytes that are not found in modern wheat, some of which belong to taxa with known beneficial effects.

Ejaculated mammalian spermatozoa must reside in the female genital tract for some time before gaining the ability to fertilize the egg. During this time, spermatozoa undergo some physiological changes that collectively are called capacitation. Capacitation of mammalian spermatozoa is a prerequisite for acrosome reaction, which is an exocytotic event occurring before fertilization. The specific biophysical and biochemical changes that accompany sperm capacitation and the agonists inducing acrosome reaction are not fully understood. Using SDS-gel electrophoresis and immunoblotting, we demonstrate the existence of a class of angiotensin receptors (AT1) in bovine spermatozoa. In capacitated sperm, we show that angiotensin II (ANG II) AT1 receptors are localized in the head and tail, whereas in noncapacitated cells the receptors are localized in the tail only. We find that ANG II markedly stimulates acrosomal exocytosis of capacitated bovine spermatozoa in vitro in a concentration range of 0.1–10 nM. No effect of ANG II was found in noncapacitated cells. The ability of ANG II to stimulate the acrosome reaction depends on the presence of calcium ions in the incubation medium. The ANG II-induced acrosome reaction was markedly inhibited by a selective AT1 receptor antagonist, losartan (DUP 753). PD-123319, a selective antagonist of the ANG II AT2 receptor, had no effect on the ANG II-induced acrosome reaction. Thus ANG II via activation of AT1 receptors may play a regulatory role in the induction of the acrosome reaction.

The measurement of disease development is integral in studies on plant-microbe interactions. To address the need for a dynamic and quantitative disease evaluation, we developed PathTrack , and used it to analyse the interaction of plants with Botrytis cinerea. PathTrack is composed of an infection chamber, a photography unit and software that produces video files and numerical values of disease progression. We identified a previously unrecognized infection stage and determined numerical parameters of pathogenic development. Using these parameters, we identified differences in disease dynamics between seemingly similar B. cinerea pathogenicity mutants, and revealed new details on plant susceptibility to the fungus. We showed that the difference between the lesion expansion rate on leaves and colony spreading rate on artificial medium reflects the levels of the plant immune system, suggesting that this parameter can be used to quantify plant defence. Our results shed new light and reveal new details of the interaction between the model necrotrophic pathogen B. cinerea and plants. The concept that we present is universal and may be applied to facilitate the study of various types of plant-pathogen association.