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1.  Production of cecropin A antimicrobial peptide in rice seed endosperm 
BMC Plant Biology  2014;14:102.
Cecropin A is a natural antimicrobial peptide that exhibits rapid, potent and long-lasting lytic activity against a broad spectrum of pathogens, thus having great biotechnological potential. Here, we report a system for producing bioactive cecropin A in rice seeds.
Transgenic rice plants expressing a codon-optimized synthetic cecropin A gene drived by an endosperm-specific promoter, either the glutelin B1 or glutelin B4 promoter, were generated. The signal peptide sequence from either the glutelin B1 or the glutelin B4 were N-terminally fused to the coding sequence of the cecropin A. We also studied whether the presence of the KDEL endoplasmic reticulum retention signal at the C-terminal has an effect on cecropin A subcellular localization and accumulation. The transgenic rice plants showed stable transgene integration and inheritance. We show that cecropin A accumulates in protein storage bodies in the rice endosperm, particularly in type II protein bodies, supporting that the glutelin N-terminal signal peptides play a crucial role in directing the cecropin A to this organelle, independently of being tagged with the KDEL endoplasmic reticulum retention signal. The production of cecropin A in transgenic rice seeds did not affect seed viability or seedling growth. Furthermore, transgenic cecropin A seeds exhibited resistance to infection by fungal and bacterial pathogens (Fusarium verticillioides and Dickeya dadantii, respectively) indicating that the in planta-produced cecropin A is biologically active.
Rice seeds can sustain bioactive cecropin A production and accumulation in protein bodies. The system might benefit the production of this antimicrobial agent for subsequent applications in crop protection and food preservation.
PMCID: PMC4032361  PMID: 24755305
Rice; Antimicrobial peptides; Cecropin A; Endosperm; Protein bodies; Pathogen resistance; Fusarium verticillioides; Dickeya dadantii; Oryza sativa
2.  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.
PMCID: PMC4298167  PMID: 25646121
cell number; image analysis software; rice; root; tissue area; transverse histological section; histological phenotype scoring
3.  International Consortium of Rice Mutagenesis: resources and beyond 
Rice  2013;6(1):39.
Rice is one of the most important crops in the world. The rice community needs to cooperate and share efforts and resources so that we can understand the functions of rice genes, especially those with a role in important agronomical traits, for application in agricultural production. Mutation is a major source of genetic variation that can be used for studying gene function. We will present here the status of mutant collections affected in a random manner by physical/chemical and insertion mutageneses.
As of early September 2013, a total of 447, 919 flanking sequence tags from rice mutant libraries with T-DNA, Ac/Ds, En/Spm, Tos17, nDART/aDART insertions have been collected and publicly available. From these, 336,262 sequences are precisely positioned on the japonica rice chromosomes, and 67.5% are in gene interval. We discuss the genome coverage and preference of the insertion, issues limiting the exchange and use of the current collections, as well as new and improved resources. We propose a call to renew all mutant populations as soon as possible. We also suggest that a common web portal should be established for ordering seeds.
PMCID: PMC3946042  PMID: 24341871
Functional genomics; Mutants; Rice
4.  Transcription factor OsHsfC1b regulates salt tolerance and development in Oryza sativa ssp. japonica 
AoB Plants  2012;2012:pls011.
The paper describes the functional analysis of a class C heat shock transcription factor from rice (Oryza sativa). OsHsfC1b is shown to play a role in ABA-mediated salt stress tolerance and is required for plant growth under non-stress conditions.
Background and aims
Salt stress leads to attenuated growth and productivity in rice. Transcription factors like heat shock factors (HSFs) represent central regulators of stress adaptation. Heat shock factors of the classes A and B are well established as regulators of thermal and non-thermal stress responses in plants; however, the role of class C HSFs is unknown. Here we characterized the function of the OsHsfC1b (Os01g53220) transcription factor from rice.
We analysed the expression of OsHsfC1b in the rice japonica cultivars Dongjin and Nipponbare exposed to salt stress as well as after mannitol, abscisic acid (ABA) and H2O2 treatment. For functional characterization of OsHsfC1b, we analysed the physiological response of a T-DNA insertion line (hsfc1b) and two artificial micro-RNA (amiRNA) knock-down lines to salt, mannitol and ABA treatment. In addition, we quantified the expression of small Heat Shock Protein (sHSP) genes and those related to signalling and ion homeostasis by quantitative real-time polymerase chain reaction in roots exposed to salt. The subcellular localization of OsHsfC1b protein fused to green fluorescent protein (GFP) was determined in Arabidopsis mesophyll cell protoplasts.
Principal results
Expression of OsHsfC1b was induced by salt, mannitol and ABA, but not by H2O2. Impaired function of OsHsfC1b in the hsfc1b mutant and the amiRNA lines led to decreased salt and osmotic stress tolerance, increased sensitivity to ABA, and temporal misregulation of salt-responsive genes involved in signalling and ion homeostasis. Furthermore, sHSP genes showed enhanced expression in knock-down plants under salt stress. We observed retarded growth of hsfc1b and knock-down lines in comparison with control plants under non-stress conditions. Transient expression of OsHsfC1b fused to GFP in protoplasts revealed nuclear localization of the transcription factor.
OsHsfC1b plays a role in ABA-mediated salt stress tolerance in rice. Furthermore, OsHsfC1b is involved in the response to osmotic stress and is required for plant growth under non-stress conditions.
PMCID: PMC3357053  PMID: 22616023
5.  Leucine-Rich repeat receptor kinases are sporadically distributed in eukaryotic genomes 
Plant leucine-rich repeat receptor-like kinases (LRR-RLKs) are receptor kinases that contain LRRs in their extracellular domain. In the last 15 years, many research groups have demonstrated major roles played by LRR-RLKs in plants during almost all developmental processes throughout the life of the plant and in defense/resistance against a large range of pathogens. Recently, a breakthrough has been made in this field that challenges the dogma of the specificity of plant LRR-RLKs.
We analyzed ~1000 complete genomes and show that LRR-RK genes have now been identified in 8 non-plant genomes. We performed an exhaustive phylogenetic analysis of all of these receptors, revealing that all of the LRR-containing receptor subfamilies form lineage-specific clades. Our results suggest that the association of LRRs with RKs appeared independently at least four times in eukaryotic evolutionary history. Moreover, the molecular evolutionary history of the LRR-RKs found in oomycetes is reminiscent of the pattern observed in plants: expansion with amplification/deletion and evolution of the domain organization leading to the functional diversification of members of the gene family. Finally, the expression data suggest that oomycete LRR-RKs may play a role in several stages of the oomycete life cycle.
In view of the key roles that LRR-RLKs play throughout the entire lifetime of plants and plant-environment interactions, the emergence and expansion of this type of receptor in several phyla along the evolution of eukaryotes, and particularly in oomycete genomes, questions their intrinsic functions in mimicry and/or in the coevolution of receptors between hosts and pathogens.
PMCID: PMC3268121  PMID: 22185365
6.  Transcript profiling of crown rootless1 mutant stem base reveals new elements associated with crown root development in rice 
BMC Genomics  2011;12:387.
In rice, the major part of the post-embryonic root system is made of stem-derived roots named crown roots (CR). Among the few characterized rice mutants affected in root development, crown rootless1 mutant is unable to initiate crown root primordia. CROWN ROOTLESS1 (CRL1) is induced by auxin and encodes an AS2/LOB-domain transcription factor that acts upstream of the gene regulatory network controlling CR development.
To identify genes involved in CR development, we compared global gene expression profile in stem bases of crl1 mutant and wild-type (WT) plants. Our analysis revealed that 250 and 236 genes are down- and up-regulated respectively in the crl1 mutant. Auxin induces CRL1 expression and consequently it is expected that auxin also alters the expression of genes that are early regulated by CRL1. To identify genes under the early control of CRL1, we monitored the expression kinetics of a selected subset of genes, mainly chosen among those exhibiting differential expression, in crl1 and WT following exogenous auxin treatment. This analysis revealed that most of these genes, mainly related to hormone, water and nutrient, development and homeostasis, were likely not regulated directly by CRL1. We hypothesized that the differential expression for these genes observed in the crl1 mutant is likely a consequence of the absence of CR formation. Otherwise, three CRL1-dependent auxin-responsive genes: FSM (FLATENNED SHOOT MERISTEM)/FAS1 (FASCIATA1), GTE4 (GENERAL TRANSCRIPTION FACTOR GROUP E4) and MAP (MICROTUBULE-ASSOCIATED PROTEIN) were identified. FSM/FAS1 and GTE4 are known in rice and Arabidopsis to be involved in the maintenance of root meristem through chromatin remodelling and cell cycle regulation respectively.
Our data showed that the differential regulation of most genes in crl1 versus WT may be an indirect consequence of CRL1 inactivation resulting from the absence of CR in the crl1 mutant. Nevertheless some genes, FAS1/FSM, GTE4 and MAP, require CRL1 to be induced by auxin suggesting that they are likely directly regulated by CRL1. These genes have a function related to polarized cell growth, cell cycle regulation or chromatin remodelling. This suggests that these genes are controlled by CRL1 and involved in CR initiation in rice.
PMCID: PMC3163228  PMID: 21806801
7.  Identification of precursor transcripts for 6 novel miRNAs expands the diversity on the genomic organisation and expression of miRNA genes in rice 
BMC Plant Biology  2008;8:123.
The plant miRNAs represent an important class of endogenous small RNAs that guide cleavage of an mRNA target or repress its translation to control development and adaptation to stresses. MiRNAs are nuclear-encoded genes transcribed by RNA polymerase II, producing a primary precursor that is subsequently processed by DCL1 an RNase III Dicer-like protein.
In rice hundreds of miRNAs have been described or predicted, but little is known on their genes and precursors which are important criteria to distinguish them from siRNAs. Here we develop a combination of experimental approaches to detect novel miRNAs in rice, identify their precursor transcripts and genes and predict or validate their mRNA targets.
We produced four cDNA libraries from small RNA fractions extracted from distinct rice tissues. By in silico analysis we selected 6 potential novel miRNAs, and confirmed that their expression requires OsDCL1. We predicted their targets and used 5'RACE to validate cleavage for three of them, targeting a PPR, an SPX domain protein and a GT-like transcription factor respectively.
In addition, we identified precursor transcripts for the 6 miRNAs expressed in rice, showing that these precursors can be efficiently processed using a transient expression assay in transfected Nicotiana benthamiana leaves. Most interestingly, we describe two precursors producing tandem miRNAs, but in distinct arrays. We focus on one of them encoding osa-miR159a.2, a novel miRNA produced from the same stem-loop structure encoding the conserved osa-miR159a.1. We show that this dual osa-miR159a.2-osa-miR159a.1 structure is conserved in distant rice species and maize. Finally we show that the predicted mRNA target of osa-miR159a.2 encoding a GT-like transcription factor is cleaved in vivo at the expected site.
The combination of approaches developed here identified six novel miRNAs expressed in rice which can be clearly distinguished from siRNAs. Importantly, we show that two miRNAs can be produced from a single precursor, either from tandem stem-loops or tandemly arrayed in a single stem-loop. This suggests that processing of these precursors could be an important regulatory step to produce one or more functional miRNAs in plants and perhaps coordinate cleavage of distinct targets in the same plant tissue.
PMCID: PMC2607281  PMID: 19055717
8.  Oryza Tag Line, a phenotypic mutant database for the Génoplante rice insertion line library 
Nucleic Acids Research  2007;36(Database issue):D1022-D1027.
To organize data resulting from the phenotypic characterization of a library of 30 000 T-DNA enhancer trap (ET) insertion lines of rice (Oryza sativa L cv. Nipponbare), we developed the Oryza Tag Line (OTL) database ( OTL structure facilitates forward genetic search for specific phenotypes, putatively resulting from gene disruption, and/or for GUSA or GFP reporter gene expression patterns, reflecting ET-mediated endogenous gene detection. In the latest version, OTL gathers the detailed morpho-physiological alterations observed during field evaluation and specific screens in a first set of 13 928 lines. Detection of GUS or GFP activity in specific organ/tissues in a subset of the library is also provided. Search in OTL can be achieved through trait ontology category, organ and/or developmental stage, keywords, expression of reporter gene in specific organ/tissue as well as line identification number. OTL now contains the description of 9721 mutant phenotypic traits observed in 2636 lines and 1234 GUS or GFP expression patterns. Each insertion line is documented through a generic passport data including production records, seed stocks and FST information. 8004 and 6101 of the 13 928 lines are characterized by at least one T-DNA and one Tos17 FST, respectively that OTL links to the rice genome browser OryGenesDB.
PMCID: PMC2238859  PMID: 17947330

Results 1-8 (8)