Culture Degeneration Reduces Sex-Related Gene Expression, Alters Metabolite Production and Reduces Insect Pathogenic Response in Cordyceps militaris.

Cordyceps militaris is an entomopathogenic ascomycete, known primarily for infecting lepidopteran larval (caterpillars) and pupal hosts. Cordycepin, a secondary metabolite produced by this fungus has anti-inflammatory properties and other pharmacological activities. However, little is known about the biological role of this adenosine derivate and its stabilising compound pentostatin in the context of insect infection the life cycle of C. militaris. During repeated subcultivation under laboratory conditions a degeneration of C. militaris marked by decreasing levels of cordycepin production can occur. Here, using degenerated and parental control strains of an isolate of C. militaris, we found that lower cordycepin production coincides with the decline in the production of various other metabolites as well as the reduced expression of genes related to sexual development. Additionally, infection of Galleria mellonella (greater wax moth) caterpillars indicated that cordycepin inhibits the immune response in host haemocytes. Accordingly, the pathogenic response to the degenerated strain was reduced. These data indicate that there are simultaneous changes in sexual reproduction, secondary metabolite production, insect immunity and infection by C. militaris. This study may have implications for biological control of insect crop pests by fungi.

Mode of Action of the Catalytic Site in the N-Terminal Ribosome-Inactivating Domain of JIP60.

Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein (RIP) from barley (Hordeum vulgare) and is involved in the plant immune response dependent on jasmonate hormones. Here, we demonstrate in Nicotiana benthamiana that transient expression of the N-terminal domain of JIP60, from which the inhibitor domain (amino acids 163-185) is removed, initiates cell death, leading to extensive necrosis of leaf tissues. We used structure prediction of JIP60 to identify potential catalytic amino acids in the active site and tested these by mutagenesis and in planta assays of necrosis induction by expression in N. benthamiana, as well as through an in vitro translation-inactivation assay. We found that Tyr 96, Glu 201, Arg 204, and Trp 234 in the presumptive active site of JIP60 are conserved in 815 plant RIPs in the Pfam database that were identified by HUMMR as containing a RIP domain. When these amino acid residues are individually mutated, the necrosis-inducing activity is completely abolished. We therefore propose that the role of these amino acids in JIP60 activity is to depurinate adenosine in ribosomes. This study provides insight into the catalytic mechanism of JIP60.

Consequences of symbiont co-infections for insect host phenotypes.

Most animals host communities of symbiotic bacteria. In insects, these symbionts may have particularly intimate interactions with their hosts: many are intracellular and can play important roles in host ecology and evolution, including protection against natural enemies. We investigated how interactions between different species or strains of endosymbiotic bacteria within an aphid host influence the outcome of symbiosis for both symbiont and host. We first asked whether different combinations of facultative symbiont species or strains can exist in stable co-infections. We then investigated whether the benefits that facultative bacteria confer on their hosts (protection against natural enemies) are enhanced, reduced or unaltered by the presence of a co-infecting symbiont. We asked this both for co-infecting symbionts that confer different phenotypes on their hosts (protection against fungal pathogens vs. parasitoid wasps) and symbionts with overlapping functions. Finally, we investigated the additional survival costs to aphids of carrying multiple infections of symbiont species or strains, and compared symbiont titres in double and single infections. We found that stable co-infections were possible between all of the combinations of facultative symbiont species (Regiella insecticola + Hamiltonella defensa, Regiella + Rickettsiella sp., Regiella + Spiroplasma sp.) and strains (Hamiltonella) that we studied. Where symbionts provided protection against different natural enemies, no alteration in protection was observed in the presence of co-infections. Where symbionts provided protection against the same natural enemy, the level of protection corresponded to the higher of the two symbionts present. In some instances, aphid hosts suffered additional survival costs when hosting double infections. In the case of Hamiltonella, however, infection with multiple strains of the same symbiont led to lower symbiont titres than single infections, and actually improved aphid survival. We conclude that the long-term maintenance of symbiont co-infections in aphids is likely to be determined primarily by costs of co-infections and in some instances by redundancy of symbiont benefits.

Unravelling mycorrhiza-induced wheat susceptibility to the English grain aphid Sitobion avenae.

Arbuscular mycorrhizal (AM) fungi are root symbionts that can increase or decrease aphid growth rates and reproduction, but the reason by which this happens is unknown. To investigate the underlying mechanisms of this interaction, we examined the effect of AM fungi on the English Grain aphid (Sitobion avenae) development, reproduction, attraction, settlement and feeding behaviour on two naturally susceptible varieties Triticum aestivum (L.) variety Solstice and T. monococcum MDR037, and two naturally resistant lines, T. monococcum MDR045 and MDR049. Mycorrhizal colonisation increased the attractiveness of T. aestivum var. Solstice to aphids, but there was no effect on aphid development on this variety. Using the Electrical Penetration Graph (EPG) technique, we found that mycorrhizal colonisation increased aphid phloem feeding on T. monococcum MDR037 and MDR045, colonisation also increased growth rate and reproductive success of S. avenae on these varieties. Mycorrhizas increased vascular bundle size, demonstrating that these fungi can influence plant anatomy. We discuss if and how this could be related to an enhanced success rate in phloem feeding in two varieties. Overall, we present and discuss how mycorrhizal fungi can affect the feeding behaviour of S. avenae in wheat, inducing susceptibility in a resistant variety.