Kentaro K. Shimizu

The wheat powdery mildew genome shows the unique evolution of an obligate biotroph

Wheat powdery mildew, Blumeria graminis forma specialis tritici, is a devastating fungal pathogen with a poorly understood evolutionary history. Here we report the draft genome sequence of wheat powdery mildew, the resequencing of three additional isolates from different geographic regions and comparative analyses with the barley powdery mildew genome. Our comparative genomic analyses identified 602 candidate effector genes, with many showing evidence of positive selection. We characterize patterns of genetic diversity and suggest that mildew genomes are mosaics of ancient haplogroups that existed before wheat domestication. The patterns of diversity in modern isolates suggest that there was no pronounced loss of genetic diversity upon formation of the new host bread wheat 10,000 years ago. We conclude that the ready adaptation of B. graminis f.sp. tritici to the new host species was based on a diverse haplotype pool that provided great genetic potential for pathogen variation.

Darwinian Selection on a Selfing Locus

The shift to self-pollination is one of the most prevalent evolutionary transitions in flowering plants. In the selfing plant Arabidopsis thaliana, pseudogenes at the SCR and SRK self-incompatibility loci are believed to underlie the evolution of self-fertilization. Positive directional selection has driven the evolutionary fixation of pseudogene alleles of SCR, leading to substantially reduced nucleotide variation. Coalescent simulations indicate that this adaptive event may have occurred very recently and is possibly associated with the post-Pleistocene expansion of A. thaliana from glacial refugia. This suggests that ancillary morphological innovations associated with self-pollination can evolve rapidly after the inactivation of the self-incompatibility response.

Robust control of the seasonal expression of the Arabidopsis FLC gene in a fluctuating environment.

Plants flower in particular seasons even in natural, fluctuating environments. The molecular basis of temperature-dependent flowering-time regulation has been extensively studied, but little is known about how gene expression is controlled in natural environments. Without a memory of past temperatures, it would be difficult for plants to detect seasons in natural, noisy environments because temperature changes occurring within a few weeks are often inconsistent with seasonal trends. Our 2-y census of the expression of a temperature-dependent flowering-time gene, AhgFLC, in a natural population of perennial Arabidopsis halleri revealed that the regulatory system of this flowering-time gene extracts seasonal cues as if it memorizes temperatures over the past 6 wk. Time-series analysis revealed that as much as 83% of the variation in the AhgFLC expression is explained solely by the temperature for the previous 6 wk, but not by the temperatures over shorter or longer periods. The accuracy of our model in predicting the gene expression pattern under contrasting temperature regimes in the transplant experiments indicates that such modeling incorporating the molecular bases of flowering-time regulation will contribute to predicting plant responses to future climate changes.

Evolution and Control of Imprinted FWA Genes in the Genus Arabidopsis

A central question in genomic imprinting is how a specific sequence is recognized as the target for epigenetic marking. In both mammals and plants, imprinted genes are often associated with tandem repeats and transposon-related sequences, but the role of these elements in epigenetic gene silencing remains elusive. FWA is an imprinted gene in Arabidopsis thaliana expressed specifically in the female gametophyte and endosperm. Tissue-specific and imprinted expression of FWA depends on DNA methylation in the FWA promoter, which is comprised of two direct repeats containing a sequence related to a SINE retroelement. Methylation of this element causes epigenetic silencing, but it is not known whether the methylation is targeted to the SINE-related sequence itself or the direct repeat structure is also necessary. Here we show that the repeat structure in the FWA promoter is highly diverse in species within the genus Arabidopsis. Four independent tandem repeat formation events were found in three closely related species. Another related species, A. halleri, did not have a tandem repeat in the FWA promoter. Unexpectedly, even in this species, FWA expression was imprinted and the FWA promoter was methylated. In addition, our expression analysis of FWA gene in vegetative tissues revealed high frequency of intra-specific variation in the expression level. In conclusion, we show that the tandem repeat structure is dispensable for the epigenetic silencing of the FWA gene. Rather, SINE-related sequence is sufficient for imprinting, vegetative silencing, and targeting of DNA methylation. Frequent independent tandem repeat formation events in the FWA promoter led us to propose that they may be a consequence, rather than cause, of the epigenetic control. The possible significance of epigenetic variation in reproductive strategies during evolution is also discussed.

Diversity at the Mla Powdery Mildew Resistance Locus from Cultivated Barley Reveals Sites of Positive Selection

The Mla locus in barley (Hordeum vulgare) conditions isolate-specific immunity to the powdery mildew fungus (Blumeria graminis f. sp. hordei) and encodes intracellular coiled-coil (CC) domain, nucleotide-binding (NB) site, and leucine-rich repeat (LRR)-containing receptor proteins. Over the last decades, genetic studies in breeding material have identified a large number of functional resistance genes at the Mla locus. To study the structural and functional diversity of this locus at the molecular level, we isolated 23 candidate MLA cDNAs from barley accessions that were previously shown by genetic studies to harbor different Mla resistance specificities. Resistance activity was detected for 13 candidate MLA cDNAs in a transient gene-expression assay. Sequence alignment of the deduced MLA proteins improved secondary structure predictions, revealing four additional, previously overlooked LRR. Analysis of nucleotide diversity of the candidate and validated MLA cDNAs revealed 34 sites of positive selection. Recombination or gene conversion events were frequent in the first half of the gene but positive selection was also found when this region was excluded. The positively selected sites are all, except two, located in the LRR domain and cluster in predicted solvent-exposed residues of the repeats 7 to 15 and adjacent turns on the concave side of the predicted solenoid protein structure. This domain-restricted pattern of positively selected sites, together with the length conservation of individual LRR, suggests direct binding of effectors to MLA receptors.

Evolution of self-compatibility in Arabidopsis by a mutation in the male specificity gene

Ever since Darwin’s pioneering research, the evolution of self-fertilisation (selfing) has been regarded as one of the most prevalent evolutionary transitions in flowering plants. A major mechanism to prevent selfing is the self-incompatibility (SI) recognition system, which consists of male and female specificity genes at the S-locus and SI modifier genes. Under conditions that favour selfing, mutations disabling the male recognition component are predicted to enjoy a relative advantage over those disabling the female component, because male mutations would increase through both pollen and seeds whereas female mutations would increase only through seeds. Despite many studies on the genetic basis of loss of SI in the predominantly selfing plant Arabidopsis thaliana, it remains unknown whether selfing arose through mutations in the female specificity gene (S-receptor kinase, SRK), male specificity gene (S-locus cysteine-rich protein, SCR; also known as S-locus protein 11, SP11) or modifier genes, and whether any of them rose to high frequency across large geographic regions. Here we report that a disruptive 213-base-pair (bp) inversion in the SCR gene (or its derivative haplotypes with deletions encompassing the entire SCR-A and a large portion of SRK-A) is found in 95% of European accessions, which contrasts with the genome-wide pattern of polymorphism in European A. thaliana. Importantly, interspecific crossings using Arabidopsis halleri as a pollen donor reveal that some A. thaliana accessions, including Wei-1, retain the female SI reaction, suggesting that all female components including SRK are still functional. Moreover, when the 213-bp inversion in SCR was inverted and expressed in transgenic Wei-1 plants, the functional SCR restored the SI reaction. The inversion within SCR is the first mutation disrupting SI shown to be nearly fixed in geographically wide samples, and its prevalence is consistent with theoretical predictions regarding the evolutionary advantage of mutations in male components.

Using knockout mutants to reveal the growth costs of defensive traits

We used a selection of Arabidopsis thaliana mutants with knockouts in defence genes to demonstrate growth costs of trichome development and glucosinolate production. Four of the seven defence mutants had significantly higher size-standardized growth rates (SGRs) than the wild-type in early life, although this benefit declined as plants grew larger. SGR is known to be a good predictor of success under high-density conditions, and we confirmed that mutants with higher growth rates had a large advantage when grown in competition. Despite the lack of differences in flowering-time genes, the mutants differed in flowering time, a trait that strongly correlated with early growth rate. Aphid herbivory decreased plant growth rate and increased flowering time, and aphid population growth rate was closely coupled to the growth rate of the host plant. Small differences in early SGR thus had cascading effects on both flowering time and herbivore populations.

The allopolyploid Arabidopsis kamchatica originated from multiple individuals of Arabidopsis lyrata and Arabidopsis halleri

Polyploidization, or genome duplication, has played a critical role in the diversification of animals, fungi and plants. Little is known about the population structure and multiple origins of polyploid species because of the difficulty in identifying multiple homeologous nuclear genes. The allotetraploid species Arabidopsis kamchatica is closely related to the model species Arabidopsis thaliana and is distributed in a broader climatic niche than its parental species. Here, we performed direct sequencing of homeologous pairs of the low‐copy nuclear genes WER and CHS by designing homeolog‐specific primers, and obtained also chloroplast and ribosomal internal transcribed spacer sequences. Phylogenetic analysis showed that 50 individuals covering the distribution range including North America are allopolyploids derived from Arabidopsis lyrata and Arabidopsis halleri. Three major clusters within A. kamchatica were detected using Bayesian clustering. One cluster has widespread distribution. The other two are restricted to the southern part of the distribution range including Japan, where the parent A. lyrata is not currently distributed. This suggests that the mountains in Central Honshu and surrounding areas in Japan served as refugia during glacial–interglacial cycles and retained this diversity. We also found that multiple haplotypes of nuclear and chloroplast sequences of A. kamchatica are identical to those of their parental species. This indicates that multiple diploid individuals contributed to the origin of A. kamchatica. The haplotypes of low‐copy nuclear genes in Japan suggest independent polyploidization events rather than introgression. Our findings suggest that self‐compatibility and gene silencing occurred independently in different origins.

Independent origins of self-compatibility in Arabidopsis thaliana

The evolution from outcrossing based on self‐incompatibility (SI) to a selfing system is one of the most prevalent transitions in flowering plants. It has been suggested that the loss of SI in Arabidopsis thaliana is associated with pseudogene formation at the SCR male component of the S locus. Recent work, however, suggests that alternative alleles with large deletions at the S locus are also present and may be responsible for the evolution of self‐compatibility in this species. We demonstrate that most of these deletion alleles are evolutionarily derived from an S haplotype (haplogroups A) that already possessed the SCR pseudogene. This haplotype and its deletion variants are nearly fixed in Europe. Together with previous transgenic data, these results suggest that the pseudogenization of ΨSCR1 gene changed the SI phenotype in the majority of A. thaliana accessions, and was a critical step in the evolution of selfing in this species. Two other haplogroups (B and C) were also identified, the former of which contains a novel and possibly functional SCR allele. In contrast to haplogroups A, these two haplogroups are found primarily in Africa and Asia. These results suggest that self‐compatibility, which appears to be fixed in this species, arose multiple times with different genetic bases, and indicates that a species‐specific trait is associated with parallel evolution at the molecular level.

MAA3 (MAGATAMA3) helicase gene is required for female gametophyte development and pollen tube guidance in Arabidopsis thaliana.

The female gametophyte plays a central role in the sexual reproduction of angiosperms. We previously isolated the maa3 (magatama3) mutant of Arabidopsis thaliana, defective in development of the female gametophyte, micropylar pollen tube guidance, and preventing the attraction of multiple pollen tubes. We here observed that the nucleolus of polar nuclei is small, and that the fusion of polar nuclei often did not occur at the time of pollination. The MAA3 gene encodes a homolog of yeast Sen1 helicase, required for RNA metabolism. It is suggested that MAA3 may regulate RNA molecules responsible for nucleolar organization and pollen tube guidance.