Molecular characterization reveals no functional evidence for naturally occurring cross-kingdom RNA interference in the early stages of Botrytis cinerea-tomato interaction.

Plant immune responses are triggered during the interaction with pathogens. The fungus Botrytis cinerea has previously been reported to use small RNAs (sRNAs) as effector molecules capable of interfering with the host immune response. Conversely, a host plant produces sRNAs that may interfere with the infection mechanism of an intruder. We used high-throughput sequencing to identify sRNAs produced by B. cinerea and Solanum lycopersicum (tomato) during early phases of interaction and to examine the expression of their predicted mRNA targets in the other organism. A total of 7042 B. cinerea sRNAs were predicted to target 3185 mRNAs in tomato. Of the predicted tomato target genes, 163 were indeed transcriptionally down-regulated during the early phase of infection. Several experiments were performed to study a causal relation between the production of B. cinerea sRNAs and the down-regulation of predicted target genes in tomato. We generated B. cinerea mutants in which a transposon region was deleted that is the source of c.10% of the fungal sRNAs. Furthermore, mutants were generated in which both Dicer-like genes (Bcdcl1 and Bcdcl2) were deleted and these displayed a >99% reduction of transposon-derived sRNA production. Neither of these mutants was significantly reduced in virulence on any plant species tested. Our results reveal no evidence for any detectable role of B. cinerea sRNAs in the virulence of the fungus.

Parental Expression Variation of Small RNAs Is Negatively Correlated with Grain Yield Heterosis in a Maize Breeding Population.

Heterosis refers to a quantitative phenomenon in which F1 hybrid trait values exceed the mean of the parental values in a positive direction. Generally, it is dependent on a high degree of heterozygosity, which is maintained in hybrid breeding by developing parental lines in separate, genetically distinct heterotic groups. The mobility of small RNAs (sRNAs) that mediate epigenetic regulation of gene expression renders them promising candidates for modulating the action of combined diverse genomes in trans-and evidence already indicates their contribution to transgressive phenotypes. By sequencing small RNA libraries of a panel of 21 maize parental inbred lines we found a low overlap of 35% between the sRNA populations from both distinct heterotic groups. Surprisingly, in contrast to genetic or gene expression variation, parental sRNA expression variation is negatively correlated with grain yield (GY) heterosis. Among 0.595 million expressed sRNAs, we identified 9,767, predominantly 22- and 24-nt long sRNAs, which showed an association of their differential expression between parental lines and GY heterosis of the respective hybrids. Of these sRNAs, 3,485 or 6,282 showed an association with high or low GY heterosis, respectively, thus the low heterosis associated group prevailing at 64%. The heterosis associated sRNAs map more frequently to genes that show differential expression between parental lines than reference sets. Together these findings suggest that trans-chromosomal actions of sRNAs in hybrids might add up to a negative contribution in heterosis formation, mediated by unfavorable gene expression regulation. We further revealed an exclusive accumulation of 22-nt sRNAs that are associated with low GY heterosis in pericentromeric genomic regions. That recombinational suppression led to this enrichment is indicated by its close correlation with low recombination rates. The existence of this enrichment, which we hypothesize resulted from the separated breeding of inbred lines within heterotic groups, may have implications for hybrid breeding strategies addressing the recombinational constraints characteristic of complex crop genomes.

Pollen Germination and Pollen Tube Growth of Arabidopsis thaliana: in vitro and Semi in vivo Methods.

Studies of pollen germination and post-germination development are not only essential for understanding plant reproduction but also are an excellent model system for tip-based growth. Here we describe easy, reproducible methods for germination and growth of pollen from the model plant Arabidopsis thaliana in artificial conditions. Our growth system can be used both for pollen placed directly on this artificial substrate as well as for the so-called 'semi in vivo' method. This is where a pistil is cut shortly after hand-pollination and the pollen tubes grow through the plant tissue and emerge from the cut end onto the surface of the artificial medium.

RNA 'Information Warfare' in Pathogenic and Mutualistic Interactions.

Regulatory non-coding RNAs are emerging as key players in host-pathogen interactions. Small RNAs such as microRNAs are implicated in regulating plant transcripts involved in immunity and defence. Surprisingly, RNAs with silencing properties can be translocated from plant hosts to various invading pathogens and pests. Small RNAs are now confirmed virulence factors, with the first report of fungal RNAs that travel to host cells and hijack post-transcriptional regulatory machinery to suppress host defence. Here, we argue that trans-organism movement of RNAs represents a common mechanism of control in diverse interactions between plants and other eukaryotes. We suggest that extracellular vesicles are the key to such RNA movement events. Plant pathosystems serve as excellent experimental models to dissect RNA 'information warfare' and other RNA-mediated interactions.

A novel Botrytis species is associated with a newly emergent foliar disease in cultivated Hemerocallis.

Foliar tissue samples of cultivated daylilies (Hemerocallis hybrids) showing the symptoms of a newly emergent foliar disease known as 'spring sickness' were investigated for associated fungi. The cause(s) of this disease remain obscure. We isolated repeatedly a fungal species which proved to be member of the genus Botrytis, based on immunological tests. DNA sequence analysis of these isolates, using several different phyogenetically informative genes, indicated that they represent a new Botrytis species, most closely related to B. elliptica (lily blight, fire blight) which is a major pathogen of cultivated Lilium. The distinction of the isolates was confirmed by morphological analysis of asexual sporulating cultures. Pathogenicity tests on Hemerocallis tissues in vitro demonstrated that this new species was able to induce lesions and rapid tissue necrosis. Based on this data, we infer that this new species, described here as B. deweyae, is likely to be an important contributor to the development of 'spring sickness' symptoms. Pathogenesis may be promoted by developmental and environmental factors that favour assault by this necrotrophic pathogen. The emergence of this disease is suggested to have been triggered by breeding-related changes in cultivated hybrids, particularly the erosion of genetic diversity. Our investigation confirms that emergent plant diseases are important and deserve close monitoring, especially in intensively in-bred plants.

Genome-wide meta-analysis of maize heterosis reveals the potential role of additive gene expression at pericentromeric loci.

BACKGROUND: The identification of QTL involved in heterosis formation is one approach to unravel the not yet fully understood genetic basis of heterosis - the improved agronomic performance of hybrid F1 plants compared to their inbred parents. The identification of candidate genes underlying a QTL is important both for developing markers and determining the molecular genetic basis of a trait, but remains difficult owing to the large number of genes often contained within individual QTL. To address this problem in heterosis analysis, we applied a meta-analysis strategy for grain yield (GY) of Zea mays L. as example, incorporating QTL-, hybrid field-, and parental gene expression data. RESULTS: For the identification of genes underlying known heterotic QTL, we made use of tight associations between gene expression pattern and the trait of interest, identified by correlation analyses. Using this approach genes strongly associated with heterosis for GY were discovered to be clustered in pericentromeric regions of the complex maize genome. This suggests that expression differences of sequences in recombination-suppressed regions are important in the establishment of heterosis for GY in F1 hybrids and also in the conservation of heterosis for GY across genotypes. Importantly functional analysis of heterosis-associated genes from these genomic regions revealed over-representation of a number of functional classes, identifying key processes contributing to heterosis for GY. Based on the finding that the majority of the analyzed heterosis-associated genes were addtitively expressed, we propose a model referring to the influence of cis-regulatory variation on heterosis for GY by the compensation of fixed detrimental expression levels in parents. CONCLUSIONS: The study highlights the utility of a meta-analysis approach that integrates phenotypic and multi-level molecular data to unravel complex traits in plants. It provides prospects for the identification of genes relevant for QTL, and also suggests a model for the potential role of additive expression in the formation and conservation of heterosis for GY via dominant, multigenic quantitative trait loci. Our findings contribute to a deeper understanding of the multifactorial phenomenon of heterosis, and thus to the breeding of new high yielding varieties.

Botrytis species: relentless necrotrophic thugs or endophytes gone rogue?

Plant pathology has a long-standing tradition of classifying microbes as pathogens, endophytes or saprophytes. Lifestyles of pathogens are categorized as biotrophic, necrotrophic or hemibiotrophic. Botrytis species are considered by many to be archetypal examples of necrotrophic fungi, with B. cinerea being the most extensively studied species because of its broad host range and economic impact. In this review, we discuss recent work which illustrates that B. cinerea is capable of colonizing plants internally, presumably as an endophyte, without causing any disease or stress symptoms. The extent of the facultative endophytic behaviour of B. cinerea and its relevance in the ecology and disease epidemiology may be vastly underestimated. Moreover, we discuss the recent discovery of a novel Botrytis species, B. deweyae, which normally grows as an endophyte in ornamental daylilies (Hemerocallis), but displays facultative pathogenic behaviour, and is increasingly causing economic damage. We propose that the emergence of endophytes 'gone rogue' as novel diseases may be related to increased inbreeding of hybrid lines and reduced genetic diversity. These observations lead us to argue that the sometimes inflexible classification of pathogenic microbes by their lifestyles requires serious reconsideration. There is much more variety to the interactions of Botrytis with its hosts than the eye (or the plant pathologist) can see, and this may be true for other microbes interacting with plants.

Artificial microRNAs reveal cell-specific differences in small RNA activity in pollen.

Pollen formation, while critical for the success of plant reproduction, also represents an important paradigm for differential cellular development within small groups of cells. In Arabidopsis thaliana pollen, the male meiotic product first divides asymmetrically to form a vegetative and a generative (germ) cell, the latter then dividing to generate two sperm cells. Here we have used artificial microRNAs to study small RNA processing in the different pollen cell types. Our data suggest that translational repression by small RNAs is enhanced in the sperm. This work also provides insights into germline RNA movement and the cell-autonomous action of microRNAs.

Emerging Roles for Non-Coding RNAs in Male Reproductive Development in Flowering Plants.

Knowledge of sexual reproduction systems in flowering plants is essential to humankind, with crop fertility vitally important for food security. Here, we review rapidly emerging new evidence for the key importance of non-coding RNAs in male reproductive development in flowering plants. From the commitment of somatic cells to initiating reproductive development through to meiosis and the development of pollen-containing the male gametes (sperm cells)-in the anther, there is now overwhelming data for a diversity of non-coding RNAs and emerging evidence for crucial roles for them in regulating cellular events at these developmental stages. A particularly exciting development has been the association of one example of cytoplasmic male sterility, which has become an unparalleled breeding tool for producing new crop hybrids, with a non-coding RNA locus.

Through a generation darkly: small RNAs in the gametophyte.

The various classes of small non-coding RNAs are a fundamentally important component of the transcriptome. These molecules have roles in many essential processes such as regulation of gene expression at the transcriptional and post-transcriptional levels, guidance of DNA methylation and defence against selfish replicators such as transposons. Their diversity and functions in the sporophytic generation of angiosperms is well explored compared with the gametophytic generation, where little is known about them. Recent progress in understanding their abundance, diversity and function in the gametophyte is reviewed.

Small RNA pathways are present and functional in the angiosperm male gametophyte.

Small non-coding RNAs are essential for development of the sporophyte, the somatic diploid phase of flowering plants. They are integral to key cellular processes such as defense, generation of chromatin structure, and regulation of native gene expression. Surprisingly, very little is known of their presence and function in the male haploid phase of plant development (male gametophyte/pollen grain), where dramatic cell fate changes leading to gametogenesis occur over just two mitotic divisions. We show that critical components of small RNA pathways are expressed throughout pollen development, but in a pattern that differs from the sporophyte. We also demonstrate that mature pollen accumulates a range of mature microRNAs, the class of small RNA most frequently involved in post-transcriptional regulation of endogenous gene expression. Significantly, these miRNAs cleave their target transcripts in developing pollen-a process that seemingly contributes to the purging of key regulatory transcripts from the mature pollen grain. Small RNAs are thus likely to make a hitherto unappreciated contribution to male gametophyte gene expression patterns, pollen development, and gametogenesis.

Bridging the generation gap: flowering plant gametophytes and animal germlines reveal unexpected similarities.

Alternation of generations underpins all plant life histories and is held to possess important adaptive features. A wide range of data have accumulated over the past century which suggest that alternation from sporophyte to gametophyte in angiosperms includes a significant phase of 'informational reprogramming', leaving the founder cells of the gametophyte developmentally uncommitted. This review attempts to bring together results from these historic studies with more recent data on molecular and epigenetic events which accompany alternation, gametophyte development and gametogenesis in angiosperms. It is striking that most members of the other principal group of multicellular eukaryotes--the animals--have a completely different a life history: animals generate their gametes directly from diploid germlines, often set aside early in development. Nevertheless, a comparison between animal germlines and angiosperm gametophyte development reveals a number of surprising similarities at the cytological and molecular levels. This difference in life history but similarity in developmental process is reviewed in the context of the very different life strategies adopted by plants and animals, and particularly the fact that plants do not set aside diploid germlines early in development.

MicroRNA and tasiRNA diversity in mature pollen of Arabidopsis thaliana.

BACKGROUND: New generation sequencing technology has allowed investigation of the small RNA populations of flowering plants at great depth. However, little is known about small RNAs in their reproductive cells, especially in post-meiotic cells of the gametophyte generation. Pollen - the male gametophyte - is the specialised haploid structure that generates and delivers the sperm cells to the female gametes at fertilisation. Whether development and differentiation of the male gametophyte depends on the action of microRNAs and trans-acting siRNAs guiding changes in gene expression is largely unknown. Here we have used 454 sequencing to survey the various small RNA populations present in mature pollen of Arabidopsis thaliana. RESULTS: In this study we detected the presence of 33 different microRNA families in mature pollen and validated the expression levels of 17 selected miRNAs by Q-RT-PCR. The majority of the selected miRNAs showed pollen-enriched expression compared with leaves. Furthermore, we report for the first time the presence of trans-acting siRNAs in pollen. In addition to describing new patterns of expression for known small RNAs in each of these classes, we identified 7 putative novel microRNAs. One of these, ath-MIR2939, targets a pollen-specific F-box transcript and we demonstrate cleavage of its target mRNA in mature pollen. CONCLUSIONS: Despite the apparent simplicity of the male gametophyte, comprising just two different cell types, pollen not only utilises many miRNAs and trans-acting siRNAs expressed in the somatic tissues but also expresses novel miRNAs.

Germlines: Argonautes go full cycle.

Specific Argonaute proteins and their small RNA targets are important in animal germline development. Although plants strictly do not have germlines and form their gametes from gametophytes, there is now evidence that reproductive Argonautes play equally important roles in plants.

Plants, pairing and phenotypes--two's company?

An RNA-based communication network appears to play a crucial role in regulating gene expression and in repressing viral and transposon sequences in plant genomes. In this article, we consider the evidence that gene expression might also be controlled epigenetically at a level other than non-coding RNA species-chromosome pairing. This epigenetic communication between sequences might be based--as it is in other organisms--on the physical pairing between homologues and the transfer of information between corresponding epigenetic landscapes. We suggest that paramutation might represent just one--albeit extreme and obvious--facet of a pairing-based gene expression regulation system in plants. Further exciting evidence for pairing occurring between homologues in plants is now mounting. An appreciation that pairing interactions might be important throughout plant development could assist in understanding phenomena such as endosperm imprinting, hybrid phenotypes and inbreeding depression.