Generation of unequal nuclear genotype proportions in Rhizophagus irregularis progeny causes allelic imbalance in gene transcription.

Arbuscular mycorrhizal fungi (AMF) form mutualisms with most plant species. The model AMF Rhizophagus irregularis is common in many ecosystems and naturally forms homokaryons and dikaryons. Quantitative variation in allele frequencies in clonally dikaryon offspring suggests they disproportionately inherit two distinct nuclear genotypes from their parent. This is interesting, because such progeny strongly and differentially affect plant growth. Neither the frequency and magnitude of this occurrence nor its effect on gene transcription are known. Using reduced representation genome sequencing, transcriptomics, and quantitative analysis tools, we show that progeny of homokaryons and dikaryons are qualitatively genetically identical to the parent. However, dikaryon progeny differ quantitatively due to unequal inheritance of nuclear genotypes. Allele frequencies of actively transcribed biallelic genes resembled the frequencies of the two nuclear genotypes. More biallelic genes showed transcription of both alleles than monoallelic transcription, but biallelic transcription was less likely with greater allelic divergence. Monoallelic transcription levels of biallelic genes were reduced compared with biallelic gene transcription, a finding consistent with genomic conflict. Given that genetic variation in R. irregularis is associated with plant growth, our results establish quantitative genetic variation as a future consideration when selecting AMF lines to improve plant production.

Coexistence of genetically different Rhizophagus irregularis isolates induces genes involved in a putative fungal mating response.

Arbuscular mycorrhizal fungi (AMF) are of great ecological importance because of their effects on plant growth. Closely related genotypes of the same AMF species coexist in plant roots. However, almost nothing is known about the molecular interactions occurring during such coexistence. We compared in planta AMF gene transcription in single and coinoculation treatments with two genetically different isolates of Rhizophagus irregularis in symbiosis independently on three genetically different cassava genotypes. Remarkably few genes were specifically upregulated when the two fungi coexisted. Strikingly, almost all of the genes with an identifiable putative function were known to be involved in mating in other fungal species. Several genes were consistent across host plant genotypes but more upregulated genes involved in putative mating were observed in host genotype (COL2215) compared with the two other host genotypes. The AMF genes that we observed to be specifically upregulated during coexistence were either involved in the mating pheromone response, in meiosis, sexual sporulation or were homologs of MAT-locus genes known in other fungal species. We did not observe the upregulation of the expected homeodomain genes contained in a putative AMF MAT-locus, but observed upregulation of HMG-box genes similar to those known to be involved in mating in Mucoromycotina species. Finally, we demonstrated that coexistence between the two fungal genotypes in the coinoculation treatments explained the number of putative mating response genes activated in the different plant host genotypes. This study demonstrates experimentally the activation of genes involved in a putative mating response and represents an important step towards the understanding of coexistence and sexual reproduction in these important plant symbionts.

Dual RNA-seq reveals large-scale non-conserved genotype × genotype-specific genetic reprograming and molecular crosstalk in the mycorrhizal symbiosis.

Arbuscular mycorrhizal fungi (AMF) impact plant growth and are a major driver of plant diversity and productivity. We quantified the contribution of intra-specific genetic variability in cassava (Manihot esculenta) and Rhizophagus irregularis to gene reprogramming in symbioses using dual RNA-sequencing. A large number of cassava genes exhibited altered transcriptional responses to the fungus but transcription of most of these plant genes (72%) responded in a different direction or magnitude depending on the plant genotype. Two AMF isolates displayed large differences in their transcription, but the direction and magnitude of the transcriptional responses for a large number of these genes was also strongly influenced by the genotype of the plant host. This indicates that unlike the highly conserved plant genes necessary for the symbiosis establishment, most of the plant and fungal gene transcriptional responses are not conserved and are greatly influenced by plant and fungal genetic differences, even at the within-species level. The transcriptional variability detected allowed us to identify an extensive gene network showing the interplay in plant-fungal reprogramming in the symbiosis. Key genes illustrated that the two organisms jointly program their cytoskeleton organization during growth of the fungus inside roots. Our study reveals that plant and fungal genetic variation has a strong role in shaping the genetic reprograming in response to symbiosis, indicating considerable genotype × genotype interactions in the mycorrhizal symbiosis. Such variation needs to be considered in order to understand the molecular mechanisms between AMF and their plant hosts in natural communities.

Variation in allele frequencies at the bg112 locus reveals unequal inheritance of nuclei in a dikaryotic isolate of the fungus Rhizophagus irregularis.

The genetic state of the arbuscular mycorrhizal fungus species Rhizophagus irregularis differs among isolates, including both homokaryotic and dikaryotic isolates. Via the production of multi-nucleate axexual spores, siblings of dikaryotic isolates may inherit unequal frequencies of nucleotypes. Using bg112, a microsatellite marker, previous studies revealed that lines deriving from single spores of the dikaryotic R. irregularis isolate C3 differed in their proportions of different alleles. A genomic study of single nuclei of R. irregularis, however, suggested that this marker was a multi-copy locus and that therefore it was inappropriate to study the inheritance of nuclei in dikaryotic isolates. In this study, we first analysed whole genome data of several R. irregularis isolates and demonstrated that bg112 is indeed a single copy locus in these genomes. Thus, the bg112 locus is a suitable marker to study the relative frequency of nucleotypes in R. irregularis. Second, by using amplicon sequencing, we confirmed the existence of one allele of bg112 in two homokaryotic isolates (DAOM197198 and C2) and two alleles in the dikaryotic isolate (C3). Finally, we found that the relative proportions of two bg112 alleles differed significantly among dikaryotic single-spore lines derived from isolate C3, indicating that genetically different nucleotypes are inherited unequally in this dikaryotic R. irregularis isolate.

Exposure of the mosquito vector Culex pipiens to the malaria parasite Plasmodium relictum: effect of infected blood intake on immune and antioxidant defences, fecundity and survival.

BACKGROUND: The intake of a Plasmodium-infected blood meal may affect mosquito physiology and a series of trade-offs may occur, in particular between immune defences, reproduction and self-maintenance. We evaluated the cost of exposure to Plasmodium in the mosquito vector by investigating the effect of exposure on fecundity and survival and the implication of immune and antioxidant defences in mediating this cost. METHODS: We used the natural Culex pipiens-Plasmodium relictum association. We exposed female mosquitoes to increasing levels of parasites by allowing them to feed either on uninfected canaries, Serinus canaria, (unexposed mosquitoes) or on infected canaries with low (low exposure) or high (high exposure) parasitaemia. We recorded blood meal size, fecundity (laying probability and clutch size) and survival. We quantified the expression of genes involved in immune and antioxidant defences (nitric oxide synthase, NOS; superoxide dismutase, SOD; glucose-6-phosphate dehydrogenase, G6PDH). RESULTS: We found that the laying probability of exposed females decreased with increasing exposure to the parasite and with increasing SOD expression. Clutch size of exposed females was higher compared to unexposed ones for similar blood meal size and was positively correlated to the NOS expression. We found no effect of exposure on survival. After blood meal intake, SOD increased in the three groups, NOS increased in exposed females and G6PDH increased in highly exposed females only. CONCLUSIONS: Our results illustrated a trade-off between fight against the parasite and reproduction and a cost of exposure which might be mediated by the investment in immune and/or antioxidant defences. They also showed that this trade-off could lead to opposed outcome, potentially depending on the vector physiological status. Finally, they highlighted that the ingestion of a Plasmodium-infected blood meal may affect mosquito life history traits in a complex way.