An atypical class of non-coding small RNAs is produced in rice leaves upon bacterial infection.

Non-coding small RNAs (sRNA) act as mediators of gene silencing and regulate plant growth, development and stress responses. Early insights into plant sRNAs established a role in antiviral defense and they are now extensively studied across plant-microbe interactions. Here, sRNA sequencing discovered a class of sRNA in rice (Oryza sativa) specifically associated with foliar diseases caused by Xanthomonas oryzae bacteria. Xanthomonas-induced small RNAs (xisRNAs) loci were distinctively upregulated in response to diverse virulent strains at an early stage of infection producing a single duplex of 20-22 nt sRNAs. xisRNAs production was dependent on the Type III secretion system, a major bacterial virulence factor for host colonization. xisRNA loci overlap with annotated transcripts sequences, with about half of them encoding protein kinase domain proteins. A number of the corresponding rice cis-genes have documented functions in immune signaling and xisRNA loci predominantly coincide with the coding sequence of a conserved kinase motif. xisRNAs exhibit features of small interfering RNAs and their biosynthesis depend on canonical components OsDCL1 and OsHEN1. xisRNA induction possibly mediates post-transcriptional gene silencing but they do not broadly suppress cis-genes expression on the basis of mRNA-seq data. Overall, our results identify a group of unusual sRNAs with a potential role in plant-microbe interactions.

A new comprehensive annotation of leucine-rich repeat-containing receptors in rice.

Oryza sativa (rice) plays an essential food security role for more than half of the world's population. Obtaining crops with high levels of disease resistance is a major challenge for breeders, especially today, given the urgent need for agriculture to be more sustainable. Plant resistance genes are mainly encoded by three large leucine-rich repeat (LRR)-containing receptor (LRR-CR) families: the LRR-receptor-like kinase (LRR-RLK), LRR-receptor-like protein (LRR-RLP) and nucleotide-binding LRR receptor (NLR). Using lrrprofiler, a pipeline that we developed to annotate and classify these proteins, we compared three publicly available annotations of the rice Nipponbare reference genome. The extended discrepancies that we observed for LRR-CR gene models led us to perform an in-depth manual curation of their annotations while paying special attention to nonsense mutations. We then transferred this manually curated annotation to Kitaake, a cultivar that is closely related to Nipponbare, using an optimized strategy. Here, we discuss the breakthrough achieved by manual curation when comparing genomes and, in addition to 'functional' and 'structural' annotations, we propose that the community adopts this approach, which we call 'comprehensive' annotation. The resulting data are crucial for further studies on the natural variability and evolution of LRR-CR genes in order to promote their use in breeding future resilient varieties.

Origin and Diversity of Plant Receptor-Like Kinases.

Because of their high level of diversity and complex evolutionary histories, most studies on plant receptor-like kinase subfamilies have focused on their kinase domains. With the large amount of genome sequence data available today, particularly on basal land plants and Charophyta, more attention should be paid to primary events that shaped the diversity of the RLK gene family. We thus focus on the motifs and domains found in association with kinase domains to illustrate their origin, organization, and evolutionary dynamics. We discuss when these different domain associations first occurred and how they evolved, based on a literature review complemented by some of our unpublished results.

A protocol combining multiphoton microscopy and propidium iodide for deep 3D root meristem imaging in rice: application for the screening and identification of tissue-specific enhancer trap lines.

BACKGROUND: The clear visualization of 3D organization at the cellular level in plant tissues is needed to fully understand plant development processes. Imaging tools allow the visualization of the main fluorophores and in vivo growth monitoring. Confocal microscopy coupled with the use of propidium iodide (PI) counter-staining is one of the most popular tools used to characterize the structure of root meristems in A. thaliana. However, such an approach is relatively ineffective in species with more complex and thicker root systems. RESULTS: We adapted a PI counter-staining protocol to visualize the internal 3D architecture of rice root meristems using multiphoton microscopy. This protocol is simple and compatible with the main fluorophores (CFP, GFP and mCherry). The efficiency and applicability of this protocol were demonstrated by screening a population of 57 enhancer trap lines. We successfully characterized GFP expression in all of the lines and identified 5 lines with tissue-specific expression. CONCLUSIONS: All of these resources are now available for the rice community and represent critical tools for future studies of root development.

Oak genome reveals facets of long lifespan.

Oaks are an important part of our natural and cultural heritage. Not only are they ubiquitous in our most common landscapes1 but they have also supplied human societies with invaluable services, including food and shelter, since prehistoric times2. With 450 species spread throughout Asia, Europe and America3, oaks constitute a critical global renewable resource. The longevity of oaks (several hundred years) probably underlies their emblematic cultural and historical importance. Such long-lived sessile organisms must persist in the face of a wide range of abiotic and biotic threats over their lifespans. We investigated the genomic features associated with such a long lifespan by sequencing, assembling and annotating the oak genome. We then used the growing number of whole-genome sequences for plants (including tree and herbaceous species) to investigate the parallel evolution of genomic characteristics potentially underpinning tree longevity. A further consequence of the long lifespan of trees is their accumulation of somatic mutations during mitotic divisions of stem cells present in the shoot apical meristems. Empirical4 and modelling5 approaches have shown that intra-organismal genetic heterogeneity can be selected for6 and provides direct fitness benefits in the arms race with short-lived pests and pathogens through a patchwork of intra-organismal phenotypes7. However, there is no clear proof that large-statured trees consist of a genetic mosaic of clonally distinct cell lineages within and between branches. Through this case study of oak, we demonstrate the accumulation and transmission of somatic mutations and the expansion of disease-resistance gene families in trees.

Author Correction: Rice auxin influx carrier OsAUX1 facilitates root hair elongation in response to low external phosphate.

The original version of this Article omitted the following from the Acknowledgements:'We also thank DBT-CREST BT/HRD/03/01/2002.'This has been corrected in both the PDF and HTML versions of the Article.

Rice auxin influx carrier OsAUX1 facilitates root hair elongation in response to low external phosphate.

Root traits such as root angle and hair length influence resource acquisition particularly for immobile nutrients like phosphorus (P). Here, we attempted to modify root angle in rice by disrupting the OsAUX1 auxin influx transporter gene in an effort to improve rice P acquisition efficiency. We show by X-ray microCT imaging that root angle is altered in the osaux1 mutant, causing preferential foraging in the top soil where P normally accumulates, yet surprisingly, P acquisition efficiency does not improve. Through closer investigation, we reveal that OsAUX1 also promotes root hair elongation in response to P limitation. Reporter studies reveal that auxin response increases in the root hair zone in low P environments. We demonstrate that OsAUX1 functions to mobilize auxin from the root apex to the differentiation zone where this signal promotes hair elongation when roots encounter low external P. We conclude that auxin and OsAUX1 play key roles in promoting root foraging for P in rice.

Post genomics era for orchid research.

Among 300,000 species in angiosperms, Orchidaceae containing 30,000 species is one of the largest families. Almost every habitats on earth have orchid plants successfully colonized, and it indicates that orchids are among the plants with significant ecological and evolutionary importance. So far, four orchid genomes have been sequenced, including Phalaenopsis equestris, Dendrobium catenatum, Dendrobium officinale, and Apostaceae shengen. Here, we review the current progress and the direction of orchid research in the post genomics era. These include the orchid genome evolution, genome mapping (genome-wide association analysis, genetic map, physical map), comparative genomics (especially receptor-like kinase and terpene synthase), secondary metabolomics, and genome editing.

Genome-wide association mapping for root cone angle in rice.

BACKGROUND: Plant root systems play a major role in anchoring and in water and nutrient uptake from the soil. The root cone angle is an important parameter of the root system architecture because, combined with root depth, it helps to determine the volume of soil explored by the plant. Two genes, DRO1 and SOR1, and several QTLs for root cone angle have been discovered in the last 5 years. RESULTS: To find other QTLs linked to root cone angle, a genome-wide association mapping study was conducted on two panels of 162 indica and 169 japonica rice accessions genotyped with two sets of SNP markers (genotyping-by-sequencing set with approximately 16,000 markers and high-density-rice-array set with approximately 300,000 markers). The root cone angle of all accessions was measured using a screen protractor on images taken after 1 month of plant growth in the Rhizoscope phenotyping system. The distribution of the root cone angle in the indica panel was Gaussian, but several accessions of the japonica panel (all the bulus from Indonesia and three temperate japonicas from Nepal or India) appeared as outliers with a very wide root cone angle. The data were submitted to association mapping using a mixed model with control of structure and kinship. A total of 15 QTLs for the indica panel and 40 QTLs for the japonica panel were detected. Genes underlying these QTLs (+/-50 kb from the significant markers) were analyzed. We focused our analysis on auxin-related genes, kinases, and genes involved in root developmental processes and identified 8 particularly interesting genes. CONCLUSIONS: The present study identifies new sources of wide root cone angle in rice, proposes ways to bypass some drawbacks of association mapping to further understand the genetics of the trait and identifies candidate genes deserving further investigation.

Corrigendum: New Insights on Leucine-Rich Repeats Receptor-Like Kinase Orthologous Relationships in Angiosperms.

[This corrects the article on p. 381 in vol. 8, PMID: 28424707.].

New Insights on Leucine-Rich Repeats Receptor-Like Kinase Orthologous Relationships in Angiosperms.

Leucine-Rich Repeats Receptor-Like Kinase (LRR-RLK) genes represent a large and complex gene family in plants, mainly involved in development and stress responses. These receptors are composed of an LRR-containing extracellular domain (ECD), a transmembrane domain (TM) and an intracellular kinase domain (KD). To provide new perspectives on functional analyses of these genes in model and non-model plant species, we performed a phylogenetic analysis on 8,360 LRR-RLK receptors in 31 angiosperm genomes (8 monocots and 23 dicots). We identified 101 orthologous groups (OGs) of genes being conserved among almost all monocot and dicot species analyzed. We observed that more than 10% of these OGs are absent in the Brassicaceae species studied. We show that the ECD structural features are not always conserved among orthologs, suggesting that functions may have diverged in some OG sets. Moreover, we looked at targets of positive selection footprints in 12 pairs of OGs and noticed that depending on the subgroups, positive selection occurred more frequently either in the ECDs or in the KDs.

Several wall-associated kinases participate positively and negatively in basal defense against rice blast fungus.

BACKGROUND: Receptor-like kinases are well-known to play key roles in disease resistance. Among them, the Wall-associated kinases (WAKs) have been shown to be positive regulators of fungal disease resistance in several plant species. WAK genes are often transcriptionally regulated during infection but the pathways involved in this regulation are not known. In rice, the OsWAK gene family is significantly amplified compared to Arabidopsis. The possibility that several WAKs participate in different ways to basal defense has not been addressed. Moreover, the direct requirement of rice OSWAK genes in regulating defense has not been explored. RESULTS: Here we show using rice (Oryza sativa) loss-of-function mutants of four selected OsWAK genes, that individual OsWAKs are required for quantitative resistance to the rice blast fungus, Magnaporthe oryzae. While OsWAK14, OsWAK91 and OsWAK92 positively regulate quantitative resistance, OsWAK112d is a negative regulator of blast resistance. In addition, we show that the very early transcriptional regulation of the rice OsWAK genes is triggered by chitin and is partially under the control of the chitin receptor CEBiP. Finally, we show that OsWAK91 is required for H2O2 production and sufficient to enhance defense gene expression during infection. CONCLUSIONS: We conclude that the rice OsWAK genes studied are part of basal defense response, potentially mediated by chitin from fungal cell walls. This work also shows that some OsWAKs, like OsWAK112d, may act as negative regulators of disease resistance.

Evolutionary Dynamics of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) Subfamily in Angiosperms.

Gene duplications are an important factor in plant evolution, and lineage-specific expanded (LSE) genes are of particular interest. Receptor-like kinases expanded massively in land plants, and leucine-rich repeat receptor-like kinases (LRR-RLK) constitute the largest receptor-like kinases family. Based on the phylogeny of 7,554 LRR-RLK genes from 31 fully sequenced flowering plant genomes, the complex evolutionary dynamics of this family was characterized in depth. We studied the involvement of selection during the expansion of this family among angiosperms. LRR-RLK subgroups harbor extremely contrasting rates of duplication, retention, or loss, and LSE copies are predominantly found in subgroups involved in environmental interactions. Expansion rates also differ significantly depending on the time when rounds of expansion or loss occurred on the angiosperm phylogenetic tree. Finally, using a dN/dS-based test in a phylogenetic framework, we searched for selection footprints on LSE and single-copy LRR-RLK genes. Selective constraint appeared to be globally relaxed at LSE genes, and codons under positive selection were detected in 50% of them. Moreover, the leucine-rich repeat domains, and specifically four amino acids in them, were found to be the main targets of positive selection. Here, we provide an extensive overview of the expansion and evolution of this very large gene family.

The phenome analysis of mutant alleles in Leucine-Rich Repeat Receptor-Like Kinase genes in rice reveals new potential targets for stress tolerant cereals.

Plants are constantly exposed to a variety of biotic and abiotic stresses that reduce their fitness and performance. At the molecular level, the perception of extracellular stimuli and the subsequent activation of defense responses require a complex interplay of signaling cascades, in which protein phosphorylation plays a central role. Several studies have shown that some members of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) family are involved in stress and developmental pathways. We report here a systematic analysis of the role of the members of this gene family by mutant phenotyping in the monocotyledon model plant rice, Oryza sativa. We have then targeted 176 of the ∼320 LRR-RLK genes (55.7%) and genotyped 288 mutant lines. Position of the insertion was confirmed in 128 lines corresponding to 100 LRR-RLK genes (31.6% of the entire family). All mutant lines harboring homozygous insertions have been screened for phenotypes under normal conditions and under various abiotic stresses. Mutant plants have been observed at several stages of growth, from seedlings in Petri dishes to flowering and grain filling under greenhouse conditions. Our results show that 37 of the LRR-RLK rice genes are potential targets for improvement especially in the generation of abiotic stress tolerant cereals.

The genome sequence of the orchid Phalaenopsis equestris.

Orchidaceae, renowned for its spectacular flowers and other reproductive and ecological adaptations, is one of the most diverse plant families. Here we present the genome sequence of the tropical epiphytic orchid Phalaenopsis equestris, a frequently used parent species for orchid breeding. P. equestris is the first plant with crassulacean acid metabolism (CAM) for which the genome has been sequenced. Our assembled genome contains 29,431 predicted protein-coding genes. We find that contigs likely to be underassembled, owing to heterozygosity, are enriched for genes that might be involved in self-incompatibility pathways. We find evidence for an orchid-specific paleopolyploidy event that preceded the radiation of most orchid clades, and our results suggest that gene duplication might have contributed to the evolution of CAM photosynthesis in P. equestris. Finally, we find expanded and diversified families of MADS-box C/D-class, B-class AP3 and AGL6-class genes, which might contribute to the highly specialized morphology of orchid flowers.

Immunoprofiling of Rice Root Cortex Reveals Two Cortical Subdomains.

The formation and differentiation of aerenchyma, i.e., air-containing cavities that are critical for flooding tolerance, take place exclusively in the cortex. The understanding of development and differentiation of the cortex is thus an important issue; however, studies on this tissue are limited, partly because of the lack of available molecular tools. We screened a commercially available library of cell wall antibodies to identify markers of cortical tissue in rice roots. Out of the 174 antibodies screened, eight were cortex-specific. Our analysis revealed that two types of cortical tissues are present in rice root seedlings. We named these cell layers "inner" and "outer" based on their location relative to the stele. We then used the antibodies to clarify cell identity in lateral roots. Without these markers, previous studies could not distinguish between the cortex and sclerenchyma in small lateral roots. By immunostaining lateral root sections, we showed that the internal ground tissue in small lateral roots has outer cortical identity.

The roots of future rice harvests.

Rice production faces the challenge to be enhanced by 50% by year 2030 to meet the growth of the population in rice-eating countries. Whereas yield of cereal crops tend to reach plateaus and a yield is likely to be deeply affected by climate instability and resource scarcity in the coming decades, building rice cultivars harboring root systems that can maintain performance by capturing water and nutrient resources unevenly distributed is a major breeding target. Taking advantage of gathering a community of rice root biologists in a Global Rice Science Partnership workshop held in Montpellier, France, we present here the recent progresses accomplished in this area and focal points where an international network of laboratories should direct their efforts.

PHIV-RootCell: a supervised image analysis tool for rice root anatomical parameter quantification.

We developed the PHIV-RootCell software to quantify anatomical traits of rice roots transverse section images. Combined with an efficient root sample processing method for image acquisition, this program permits supervised measurements of areas (those of whole root section, stele, cortex, and central metaxylem vessels), number of cell layers and number of cells per cell layer. The PHIV-RootCell toolset runs under ImageJ, an independent operating system that has a license-free status. To demonstrate the usefulness of PHIV-RootCell, we conducted a genetic diversity study and an analysis of salt stress responses of root anatomical parameters in rice (Oryza sativa L.). Using 16 cultivars, we showed that we could discriminate between some of the varieties even at the 6 day-olds stage, and that tropical japonica varieties had larger root sections due to an increase in cell number. We observed, as described previously, that root sections become enlarged under salt stress. However, our results show an increase in cell number in ground tissues (endodermis and cortex) but a decrease in external (peripheral) tissues (sclerenchyma, exodermis, and epidermis). Thus, the PHIV-RootCell program is a user-friendly tool that will be helpful for future genetic and physiological studies that investigate root anatomical trait variations.

Reverse genetics in rice using Tos17.

Transposon of Oryza sativa 17 (Tos17), a Ty1-Copia Class I retroelement, is one of the few active retroelements identified in rice, the main cereal crop of human consumption and the model genome for cereals. Tos17 exists in two copies in the standard Nipponbare japonica genome (n = 12 and 379 Mb). Tos17 copies are inactive in the plant grown under normal conditions. However, the copy located on chromosome 7 can be activated upon tissue culture. Plants regenerated from 3- and 5-month-old tissue cultures harbor, respectively, an average of 3.5 and 8 newly transposed copies that are stably inserted at new positions in the genome. Due to its favorable features, Tos17 has been extensively used for insertion mutagenesis of the model genome and 31,403 sequence indexed inserts harbored by regenerants/T-DNA plants are available in the databases. The corresponding seed stocks can be ordered from the laboratories which generated them. Both forward genetics and reverse genetics approaches using these lines have allowed the deciphering of gene function in rice. We report here two protocols for ascertaining the presence of a Tos17 insertion in a gene of interest among R2/T2 seeds received from Tos17 mutant stock centers: The first protocol is PCR-based and allows the identification of azygous, heterozygous and homozygous plants among progenies segregating the insertion. The second protocol is based on DNA blot analysis and can be used to identify homozygous plants carrying the Tos17 copy responsible for gene disruption while cleaning the mutant background from other unwitting mutagen inserts.