Coevolution between a Family of Parasite Virulence Effectors and a Class of LINE-1 Retrotransposons

Parasites are able to evolve rapidly and overcome host defense mechanisms, but the molecular basis of this adaptation is poorly understood. Powdery mildew fungi (Erysiphales, Ascomycota) are obligate biotrophic parasites infecting nearly 10,000 plant genera. They obtain their nutrients from host plants through specialized feeding structures known as haustoria. We previously identified the AVR _k1 powdery mildew-specific gene family encoding effectors that contribute to the successful establishment of haustoria. Here, we report the extensive proliferation of the AVR _k1 gene family throughout the genome of B. graminis, with sequences diverging in formae speciales adapted to infect different hosts. Also, importantly, we have discovered that the effectors have coevolved with a particular family of LINE-1 retrotransposons, named TE1a. The coevolution of these two entities indicates a mutual benefit to the association, which could ultimately contribute to parasite adaptation and success. We propose that the association would benefit 1) the powdery mildew fungus, by providing a mechanism for amplifying and diversifying effectors and 2) the associated retrotransposons, by providing a basis for their maintenance through selection in the fungal genome.     

Variation in Tolerance and Virulence in the Chestnut Blight Fungus-Hypovirus Interaction

Chestnut blight, caused by the fungus Cryphonectria parasitica, has been effectively controlled with double-stranded RNA hypoviruses in Europe for over 40 years. The marked reduction in the virulence of C. parasitica by hypoviruses is a phenomenon known as hypovirulence. This virus-fungus pathosystem has become a model system for the study of biological control of fungi with viruses. We studied variation in tolerance to hypoviruses in fungal hosts and variation in virulence among virus isolates from a local population in Italy. Tolerance is defined as the relative fitness of a fungal individual when infected with hypoviruses (compared to being uninfected); virulence is defined for each hypovirus as the reduction in fitness of fungal hosts relative to virus-free hosts. Six hypovirus-infected isolates of C. parasitica were sampled from the population, and each hypovirus was transferred into six hypovirus-free recipient isolates. The resulting 36 hypovirus-fungus combinations were used to estimate genetic variation in tolerance to hypoviruses, in hypovirus virulence, and in virus-fungus interactions. Four phenotypes were evaluated for each virus-fungus combination to estimate relative fitness: (i) sporulation, i.e., the number of asexual spores (conidia) produced; (ii) canker area on field-inoculated chestnut trees, (iii) vertical transmission of hypoviruses into conidia, and (iv) conidial germination. Two-way analysis of variance (ANOVA) revealed significant interactions (P < 0.001) between viruses and fungal isolates for sporulation and canker area but not for conidial germination or transmission. One-way ANOVA among hypoviruses (within each fungal isolate) and among fungal isolates (within each hypovirus) revealed significant genetic variation (P < 0.01) in hypovirus virulence and fungal tolerance within several fungal isolates, and hypoviruses, respectively. These interactions and the significant genetic variation in several fitness characters indicate the potential for future evolution of these characters. However, biological control is unlikely to break down due to evolution of tolerance to hypoviruses in the fungus because the magnitudes of tolerance and interactions were relatively small.     

The Coevolution of Secrecy and Stigmatization

We propose a coevolutionary model of secrecy and stigmatization. According to this model, secrecy functions to conceal potential fitness costs detected in oneself or one’s genetic kin. In three studies, we found that the content of participants’ distressing secrets overlapped significantly with three domains of social information that were important for inclusive fitness and served as cues for discriminating between rewarding and unrewarding interaction partners: health, mating, and social-exchange behavior. These findings support the notion that secrecy functions primarily as a defense against stigmatization by suppressing information about oneself or one’s kin that evolutionarily has been devalued in mating and social exchange.     

The RNA Chaperone Hfq Is Involved in Stress Tolerance and Virulence in Uropathogenic Proteus mirabilis

Hfq is a bacterial RNA chaperone involved in the riboregulation of diverse genes via small noncoding RNAs. Here, we show that Hfq is critical for the uropathogenic Proteus mirabilis to effectively colonize the bladder and kidneys in a murine urinary tract infection (UTI) model and to establish burned wound infection of the rats. In this regard, we found the hfq mutant induced higher IL-8 and MIF levels of uroepithelial cells and displayed reduced intra-macrophage survival. The loss of hfq affected bacterial abilities to handle H₂O₂ and osmotic pressures and to grow at 50°C. Relative to wild-type, the hfq mutant had reduced motility, fewer flagella and less hemolysin expression and was less prone to form biofilm and to adhere to and invade uroepithelial cells. The MR/P fimbrial operon was almost switched to the off phase in the hfq mutant. In addition, we found the hfq mutant exhibited an altered outer membrane profile and had higher RpoE expression, which indicates the hfq mutant may encounter increased envelope stress. With the notion of envelope disturbance in the hfq mutant, we found increased membrane permeability and antibiotic susceptibilities in the hfq mutant. Finally, we showed that Hfq positively regulated the RpoS level and tolerance to H₂O₂ in the stationary phase seemed largely mediated through the Hfq-dependent RpoS expression. Together, our data indicate that Hfq plays a critical role in P. mirabilis to establish UTIs by modulating stress responses, surface structures and virulence factors. This study suggests Hfq may serve as a scaffold molecule for development of novel anti-P. mirabilis drugs and P. mirabilis hfq mutant is a vaccine candidate for preventing UTIs.     

Two Spx Regulators Modulate Stress Tolerance and Virulence in Streptococcus suis Serotype 2

Streptococcus suis serotype 2 is an important zoonotic pathogen causing severe infections in pigs and humans. The pathogenesis of S. suis 2 infections, however, is still poorly understood. Spx proteins are a group of global regulators involved in stress tolerance and virulence. In this study, we characterized two orthologs of the Spx regulator, SpxA1 and SpxA2 in S. suis 2. Two mutant strains (ΔspxA1 and ΔspxA2) lacking the spx genes were constructed. The ΔspxA1 and ΔspxA2 mutants displayed different phenotypes. ΔspxA1 exhibited impaired growth in the presence of hydrogen peroxide, while ΔspxA2 exhibited impaired growth in the presence of SDS and NaCl. Both mutants were defective in medium lacking newborn bovine serum. Using a murine infection model, we demonstrated that the abilities of the mutant strains to colonize the tissues were significantly reduced compared to that of the wild-type strain. The mutant strains also showed a decreased level of survival in pig blood. Microarray analysis revealed a global regulatory role for SpxA1 and SpxA2. Furthermore, we demonstrated for the first time that Spx is involved in triggering the host inflammatory response. Collectively, our data suggest that SpxA1 and SpxA2 are global regulators that are implicated in stress tolerance and virulence in S. suis 2.     

Contribution of Conserved ATP-Dependent Proteases of Campylobacter jejuni to Stress Tolerance and Virulence

In prokaryotic cells the ATP-dependent proteases Lon and ClpP (Clp proteolytic subunit) are involved in the turnover of misfolded proteins and the degradation of regulatory proteins, and depending on the organism, these proteases contribute variably to stress tolerance. We constructed mutants in the lon and clpP genes of the food-borne human pathogen Campylobacter jejuni and found that the growth of both mutants was impaired at high temperature, a condition known to increase the level of misfolded protein. Moreover, the amounts of misfolded protein aggregates were increased when both proteases were absent, and we propose that both ClpP and Lon are involved in eliminating misfolded proteins in C. jejuni. In order to bind misfolded protein, ClpP has to associate with one of several Clp ATPases. Following inactivation of the ATPase genes clpA and clpX, only the clpX mutant displayed the same heat sensitivity as the clpP mutant, indicating that the ClpXP proteolytic complex is responsible for the degradation of heat-damaged proteins in C. jejuni. Notably, ClpP and ClpX are required for growth at 42°C, which is the temperature of the intestinal tract of poultry, one of the primary carriers of C. jejuni. Thus, ClpP and ClpX may be suitable targets of new intervention strategies aimed at reducing C. jejuni in poultry production. Further characterization of the clpP and lon mutants revealed other altered phenotypes, such as reduced motility, less autoagglutination, and lower levels of invasion of INT407 epithelial cells, suggesting that the proteases may contribute to the virulence of C. jejuni.     

Association between Virulence and Triazole Tolerance in the Phytopathogenic Fungus Mycosphaerella graminicola

Host resistance and synthetic antimicrobials such as fungicides are two of the main approaches used to control plant diseases in conventional agriculture. Although pathogens often evolve to overcome host resistance and antimicrobials, the majority of reports have involved qualitative host – pathogen interactions or antimicrobials targeting a single pathogen protein or metabolic pathway. Studies that consider jointly the evolution of virulence, defined as the degree of damage caused to a host by parasite infection, and antimicrobial resistance are rare. Here we compared virulence and fungicide tolerance in the fungal pathogen Mycosphaerella graminicola sampled from wheat fields across three continents and found a positive correlation between virulence and tolerance to a triazole fungicide. We also found that quantitative host resistance selected for higher pathogen virulence. The possible mechanisms responsible for these observations and their consequences for sustainable disease management are discussed.     

Calcineurin Controls Hyphal Growth,Virulence, and Drug Tolerance of Candida tropicalis

Candida tropicalis, a species closely related to Candida albicans, is an emerging fungal pathogen associated with high mortality rates of 40 to 70%. Like C. albicans and Candida dubliniensis, C. tropicalis is able to form germ tubes, pseudohyphae, and hyphae, but the genes involved in hyphal growth machinery and virulence remain unclear in C. tropicalis. Recently, echinocandin- and azole-resistant C. tropicalis isolates have frequently been isolated from various patients around the world, making treatment difficult. However, studies of the C. tropicalis genes involved in drug tolerance are limited. Here, we investigated the roles of calcineurin and its potential target, Crz1, for core stress responses and pathogenesis in C. tropicalis. We demonstrate that calcineurin and Crz1 are required for hyphal growth, micafungin tolerance, and virulence in a murine systemic infection model, while calcineurin but not Crz1 is essential for tolerance of azoles, caspofungin, anidulafungin, and cell wall-perturbing agents, suggesting that calcineurin has both Crz1-dependent and -independent functions in C. tropicalis. In addition, we found that calcineurin and Crz1 have opposite roles in controlling calcium tolerance. Calcineurin serves as a negative regulator, while Crz1 plays a positive role for calcium tolerance in C. tropicalis.     

Coevolution in temporally variable environments

Many potentially mutualistic interactions are conditional, with selection that varies between mutualism and antagonism over space and time. We develop a genetic model of temporally variable coevolution that incorporates stochastic fluctuations between mutualism and antagonism. We use this model to determine conditions necessary for the coevolution of matching traits between a host and a conditional mutualist. Using an analytical approximation, we show that matching traits will coevolve when the geometric mean interaction is mutualistic. When this condition does not hold, polymorphism and trait mismatching are maintained, and coevolutionary cycles may result. Numerical simulations verify this prediction and suggest that it remains robust in the presence of temporal autocorrelation. These results are compared with those from spatial models with unrestricted movement. The comparisons demonstrate that gene flow is unnecessary for generating empirical patterns predicted by the geographic mosaic theory of coevolution.     

Coevolution in resource-limited competition communities

A coevolutionary model of species packing is developed which allows evolutionary adjustment in both niche position and within-phenotype niche width of from one of three competing species. The environment is specified as a single resource dimension x and availability of resources along x is given by a Gaussian curve with parameters ($\text{x}\text{?}{}_{\mathrm{R}}\text{and}\text{σ}{}_{\mathrm{R}}$). The model predicts that for S species the ratio of optimal niche width (?) to σ_R is roughly independent of σ_R and can be approximated by $\text{1}\text{S}$ when the competitors are completely resource limited. Niche separation ($\text{d}\text{?}\text{w}\text{?}$) increases only moderately with increases in resource diversity σ_R and is greater for two than for three competing species. To the extent that the competitors do not utilize all resources, both optimal niche separation and niche width decrease. Many of the general trends in niche width and niche separation predicted by this coevolutionary model parallel those from optimal foraging theory and limiting similarity models of community structure. The coevolutionary model stands out, however, in the singularly high values predicted for niche separation, making coevolved communities highly invadeable. Hence, the theory suggests, as some empirical evidence indicates, that coevolved competition communities can only exist as such on remote islands or in other habitats which might be free from invasion by outside species. For such communities, resource diversity (σ_R) should have much less effect on species packing than either species number itself or the extent that these competitors are resource limited.     

Coevolution of competing species: ecological character displacement

Character displacement of competing species is studied. A model, originally developed by MacArthur and Levins (Proc. Natl. Acad. Sci. USA 51 (1964), 1207-1210) and further analyzed by Lawlor and Maynard Smith (Amer. Nat. 110 (1976), 70-99), has been reanalyzed. In the present paper, a more formally correct analysis of the MacArthur-Levins model is provided. A standard population genetics approach to sexually reproducing populations is adopted. The same conclusion as proposed by Lawlor and Maynard Smith emerges; competition can lead only to character divergence. In our analysis we either require that allopatrically evolved consumer populations must be able to coexist at an ecologically stable equilibrium (hence, we require mutual invasibility), or consider the feasibility of allopatric equilibria.     

Coevolution of physiological systems

Coevolution is the interaction in the process of evolution of different species that are closely related biologically but do not exchange genetic information. In this paper, we address the problem of coevolution of the whole organism’s physiological systems as a process of the interrelated development of structure and function as well as their regulatory systems during the formation of living organisms. We consider the coevolution of osmoregulation and the nitrogen metabolism type, systemic and individual coevolutionary strategies of cell volume regulation in poikiloosmotic and homoiosmotic animals, coevolution of effectory organs and endocrine factors in the development of water–salt homeostasis, co-involvement of neurohypophyseal nonapeptides and glucagon-like peptide-1 in the regulation of the renal function aimed at stabilizing physico-chemical parameters of extracellular fluids which make up the internal environment of the organism.     

Effects of Nutrition on Desiccation Tolerance and Virulence of Colletotrichum truncatum and Alternaria alternata Conidia

The promising mycoherbicides Colletotrichum truncatum and Alternaria alternata were grown respectively in liquid and solid semi-defined media. C. truncatum conidia produced in a medium with a C:N ratio of 5:1 showed higher desiccation tolerance (survival during storage) at 15% relative humidity and 25°C, greater germination on the host leaf and greater disease expression on Sesbania exaltata than those produced in media with C:N ratios of 15:1 or 40:1. Similar results were obtained with conidia of A. alternata produced on a medium with a C:N ratio of 15:1. Conidia washed with 0.9% (w/v) NaCl produced higher tolerance to desiccation, and greater disease incitement, than unwashed conidia of C. truncatum or conidia washed with water. In contrast, washing had no positive effect on desiccation tolerance in A. alternata .