John H. Doonan

Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.

The auxin signalling network translates dynamic input into robust patterning at the shoot apex

The plant hormone auxin is thought to provide positional information for patterning during development. It is still unclear, however, precisely how auxin is distributed across tissues and how the hormone is sensed in space and time. The control of gene expression in response to auxin involves a complex network of over 50 potentially interacting transcriptional activators and repressors, the auxin response factors (ARFs) and Aux/IAAs. Here, we perform a large‐scale analysis of the Aux/IAA‐ARF pathway in the shoot apex of Arabidopsis, where dynamic auxin‐based patterning controls organogenesis. A comprehensive expression map and full interactome uncovered an unexpectedly simple distribution and structure of this pathway in the shoot apex. A mathematical model of the Aux/IAA‐ARF network predicted a strong buffering capacity along with spatial differences in auxin sensitivity. We then tested and confirmed these predictions using a novel auxin signalling sensor that reports input into the signalling pathway, in conjunction with the published DR5 transcriptional output reporter. Our results provide evidence that the auxin signalling network is essential to create robust patterns at the shoot apex.

Silencing by plant Polycomb-group genes requires dispersed trimethylation of histone H3 at lysine 27

The plant Polycomb‐group (Pc‐G) protein CURLY LEAF (CLF) is required to repress targets such as AGAMOUS (AG) and SHOOTMERISTEMLESS (STM). Using chromatin immunoprecipitation, we identify AG and STM as direct targets for CLF and show that they carry a characteristic epigenetic signature of dispersed histone H3 lysine 27 trimethylation (H3K27me3) and localised H3K27me2 methylation. H3K27 methylation is present throughout leaf development and consistent with this, CLF is required persistently to silence AG. However, CLF is not itself an epigenetic mark as it is lost during mitosis. We suggest a model in which Pc‐G proteins are recruited to localised regions of targets and then mediate dispersed H3K27me3. Analysis of transgenes carrying AG regulatory sequences confirms that H3K27me3 can spread to novel sequences in a CLF‐dependent manner and further shows that H3K27me3 methylation is not sufficient for silencing of targets. We suggest that the spread of H3K27me3 contributes to the mitotic heritability of Pc‐G silencing, and that the loss of silencing caused by transposon insertions at plant Pc‐G targets reflects impaired spreading.

The Arabidopsis RNA-Directed DNA Methylation Argonautes Functionally Diverge Based on Their Expression and Interaction with Target Loci

This study examines the basis of functional divergence amongst the closely related Arabidopsis AGO4, AGO6, and AGO9 proteins. Using associated small RNAs, these proteins direct DNA epigenetic modifications in a tissue-specific manner. Argonaute (AGO) effectors of RNA silencing bind small RNA (sRNA) molecules and mediate mRNA cleavage, translational repression, or epigenetic DNA modification. In many organisms, these targeting mechanisms are devolved to different products of AGO multigene families. To investigate the basis of AGO functional diversification, we characterized three closely related Arabidopsis thaliana AGOs (AGO4, AGO6, and AGO9) implicated in RNA-directed DNA methylation. All three AGOs bound 5′ adenosine 24-nucleotide sRNAs, but each exhibited different preferences for sRNAs from different heterochromatin-associated loci. This difference was reduced when AGO6 and AGO9 were expressed from the AGO4 promoter, indicating that the functional diversification was partially due to differential expression of the corresponding genes. However, the AGO4-directed pattern of sRNA accumulation and DNA methylation was not fully recapitulated with AGO6 or AGO9 expressed from the AGO4 promoter. Here, we show that sRNA length and 5′ nucleotide do not account for the observed functional diversification of these AGOs. Instead, the selectivity of sRNA binding is determined by the coincident expression of the AGO and sRNA-generating loci, and epigenetic modification is influenced by interactions between the AGO protein and the different target loci. These findings highlight the importance of tissue specificity and AGO-associated proteins in influencing epigenetic modifications.

EB1 reveals mobile microtubule nucleation sites in Arabidopsis

In plants, it is unclear how dispersed cortical microtubules are nucleated, polarized and organized in the absence of centrosomes. In Arabidopsis thaliana cells, expression of a fusion between the microtubule-end-binding protein AtEB1a and green fluorescent protein (GFP) results in labelling of spindle poles, where minus ends gather. During interphase, AtEB1a–GFP labels the microtubule plus end as a comet, but also marks the minus end as a site from which microtubules can grow and shrink. These minus-end nucleation sites are mobile, explaining how the cortical array can redistribute during the cell cycle and supporting the idea of a flexible centrosome in plants.

Plant cyclins : a unified nomenclature for plant A-, B- and D-type cyclins based on sequence organization

The comparative analysis of a large number of plant cyclins of the A/B family has recently revealed that plants possess two distinct B-type groups and three distinct A-type groups of cyclins [1]. Despite earlier uncertainties, this large-scale comparative analysis has allowed an unequivocal definition of plant cyclins into either A or B classes. We present here the most important results obtained in this study, and extend them to the case of plant D-type cyclins, in which three groups are identified. For each of the plant cyclin groups, consensus sequences have been established and a new, rational, plant-wide naming system is proposed in accordance with the guidelines of the Commission on Plant Gene Nomenclature. This nomenclature is based on the animal system indicating cyclin classes by an upper-case roman letter, and distinct groups within these classes by an arabic numeral suffix. The naming of plant cyclin classes is chosen to indicate homology to their closest animal class. The revised nomenclature of all described plant cyclins is presented, with their classification into groups CycA1, CycA2, CycA3, CycB1, CycB2, CycD1, CycD2 and CycD3.

A Genetic Framework for Grain Size and Shape Variation in Wheat

Using large-scale quantitative analysis, this work reveals that grain shape and size are independent traits in both modern and primitive wheat and are under the control of distinct genetic components. Moreover, the phenotypic diversity in grain morphology found in modern commercial wheat is the result of a recent and severe bottleneck. Grain morphology in wheat (Triticum aestivum) has been selected and manipulated even in very early agrarian societies and remains a major breeding target. We undertook a large-scale quantitative analysis to determine the genetic basis of the phenotypic diversity in wheat grain morphology. A high-throughput method was used to capture grain size and shape variation in multiple mapping populations, elite varieties, and a broad collection of ancestral wheat species. This analysis reveals that grain size and shape are largely independent traits in both primitive wheat and in modern varieties. This phenotypic structure was retained across the mapping populations studied, suggesting that these traits are under the control of a limited number of discrete genetic components. We identified the underlying genes as quantitative trait loci that are distinct for grain size and shape and are largely shared between the different mapping populations. Moreover, our results show a significant reduction of phenotypic variation in grain shape in the modern germplasm pool compared with the ancestral wheat species, probably as a result of a relatively recent bottleneck. Therefore, this study provides the genetic underpinnings of an emerging phenotypic model where wheat domestication has transformed a long thin primitive grain to a wider and shorter modern grain.

Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans

Green fluorescent protein (GFP) is a useful reporter to follow the in vivo behaviour of proteins, but the wild‐type gfp gene does not function in many organisms, including many plants and filamentous fungi. We show that codon‐modified forms of gfp, produced for use in plants, function effectively in Aspergillus nidulans both as gene expression reporters and as vital reporters for protein location. To demonstrate the use of these modified gfps as reporter genes we have used fluorescence to follow ethanol‐induced GFP expression from the alcA promoter. Translational fusions with the modified gfp were used to follow protein location in living cells; plant ER‐retention signals targeted GFP to the endoplasmic reticulum, whereas fusion to the GAL4 DNA‐binding domain targeted it to the nucleus. Nuclear‐targeted GFP allowed real‐time observation of nuclear movement and division. These modified gfp genes should provide useful markers to follow gene expression, organelle behaviour and protein trafficking in real time.

G2/M-Phase–Specific Transcription during the Plant Cell Cycle Is Mediated by c-Myb–Like Transcription Factors

Plant B-type cyclin genes are expressed specifically in late G2- and M-phases during the cell cycle. Their promoters contain a common cis-acting element, called the MSA (M-specific activator) element, that is necessary and sufficient for periodic promoter activation. This motif also is present in the tobacco kinesin-like protein gene NACK1, which is expressed with timing similar to that of B-type cyclin genes. In this study, we show that G2/M-phase–specific activation of the NACK1 promoter also is regulated by the MSA element, suggesting that a defined set of G2/M-phase–specific genes are coregulated by an MSA-mediated mechanism. In a search for MSA binding factors by yeast one-hybrid screening, we identified three different Myb-like proteins that interact specifically with the MSA sequence. Unlike the majority of plant Myb-like proteins, these Myb proteins, NtmybA1, NtmybA2, and NtmybB, have three imperfect repeats in the DNA binding domain, as in animal c-Myb proteins. During the cell cycle, the level of NtmybB mRNA did not change significantly, whereas the levels of NtmybA1 and A2 mRNAs fluctuated and peaked at M-phase, when B-type cyclin genes were maximally induced. In transient expression assays, NtmybA1 and A2 activated the MSA-containing promoters, whereas NtmybB repressed them. Furthermore, expression of NtmybB repressed the transcriptional activation mediated by NtmybA2. Our data show that a group of plant Myb proteins that are structurally similar to animal c-Myb proteins have unexpected roles in G2/M-phase by modulating the expression of B-type cyclin genes and may regulate a suite of coexpressed genes.

Patterns of cell division revealed by transcriptional regulation of genes during the cell cycle in plants

Transcripts from five cell cycle related genes accumulate in isolated cells dispersed throughout the actively dividing regions of plant meristems. We propose that this pattern reflects gene expression during particular phases of the cell division cycle. The high proportion of isolated cells suggests that synchrony between daughter cells is rapidly lost following mitosis. This is the first time that such a cell specific expression pattern has been described in a higher organism. Counterstaining with a DNA specific dye revealed that transcripts from three genes (two mitotic cyclins and a cdc2‐like gene) accumulate during part of interphase and early mitosis whereas transcripts from a histone H4 gene are preferentially detected only in interphase cells. Double labelling for cyclin and histone H4 transcripts confirms that these genes are expressed in different cells, and therefore at different phases of the cell cycle. The results suggest that transcriptional regulation of cell cycle related genes may be important in controlling cell division in plants, and that these genes are useful markers for identifying cells at specific phases of the cell cycle within plant meristems.