CROWN ROOTLESS1 binds DNA with a relaxed specificity and activates OsROP and OsbHLH044 genes involved in crown root formation in rice.

In cereals, the root system is mainly composed of post-embryonic shoot-borne roots, named crown roots. The CROWN ROOTLESS1 (CRL1) transcription factor, belonging to the ASYMMETRIC LEAVES2-LIKE/LATERAL ORGAN BOUNDARIES DOMAIN (ASL/LBD) family, is a key regulator of crown root initiation in rice (Oryza sativa). Here, we show that CRL1 can bind, both in vitro and in vivo, not only the LBD-box, a DNA sequence recognized by several ASL/LBD transcription factors, but also another not previously identified DNA motif that was named CRL1-box. Using rice protoplast transient transactivation assays and a set of previously identified CRL1-regulated genes, we confirm that CRL1 transactivates these genes if they possess at least a CRL1-box or an LBD-box in their promoters. In planta, ChIP-qPCR experiments targeting two of these genes that include both a CRL1- and an LBD-box in their promoter show that CRL1 binds preferentially to the LBD-box in these promoter contexts. CRISPR/Cas9-targeted mutation of these two CRL1-regulated genes, which encode a plant Rho GTPase (OsROP) and a basic helix-loop-helix transcription factor (OsbHLH044), show that both promote crown root development. Finally, we show that OsbHLH044 represses a regulatory module, uncovering how CRL1 regulates specific processes during crown root formation.

Efficient Genome Editing in Rice Protoplasts Using CRISPR/CAS9 Construct.

Genome editing technologies, mainly CRISPR/CAS9, are revolutionizing plant biology and breeding. Since the demonstration of its effectiveness in eukaryotic cells, a very large number of derived technologies has emerged. Demonstrating and comparing the effectiveness of all these new technologies in entire plants is a long, tedious, and labor-intensive process that generally involves the production of transgenic plants and their analysis. Protoplasts, plant cells free of their walls, offer a simple, high-throughput system to test the efficiency of these editing technologies in a few weeks' time span. We have developed a routine protocol using protoplasts to test editing technologies in rice. Our protocol allows to test more than 30 constructs in protoplasts prepared from leaf tissues of 100, 9-11-day-old seedlings. CRISPR/CAS9 construct effectiveness can be clearly established within less than a week. We provide here a full protocol, from designing sgRNA to mutation analysis.

Beyond Seek and Destroy: how to Generate Allelic Series Using Genome Editing Tools.

Genome editing tools have greatly facilitated the functional analysis of genes of interest by targeted mutagenesis. Many usable genome editing tools, including different site-specific nucleases and editor databases that allow single-nucleotide polymorphisms (SNPs) to be introduced at a given site, are now available. These tools can be used to generate high allelic diversity at a given locus to facilitate gene function studies, including examining the role of a specific protein domain or a single amino acid. We compared the effects, efficiencies and mutation types generated by our LbCPF1, SpCAS9 and base editor (BECAS9) constructs for the OsCAO1 gene. SpCAS9 and LbCPF1 have similar efficiencies in generating mutations but differ in the types of mutations induced, with the majority of changes being single-nucleotide insertions and short deletions for SpCAS9 and LbCPF1, respectively. The proportions of heterozygotes also differed, representing a majority in our LbCPF1, while with SpCAS9, we obtained a large number of biallelic mutants. Finally, we demonstrated that it is possible to specifically introduce stop codons using the BECAS9 with an acceptable efficiency of approximately 20%. Based on these results, a rational choice among these three alternatives may be made depending on the type of mutation that one wishes to introduce, the three systems being complementary. SpCAS9 remains the best choice to generate KO mutations in primary transformants, while if the desired gene mutation interferes with regeneration or viability, the use of our LbCPF1 construction will be preferred, because it produces mainly heterozygotes. LbCPF1 has been described in other studies as being as effective as SpCAS9 in generating homozygous and biallelic mutations. It will remain to be clarified in the future, whether the different LbCFP1 constructions have different efficiencies and determine the origin of these differences. Finally, if one wishes to specifically introduce stop codons, BECAS9 is a viable and efficient alternative, although it has a lower efficiency than SpCAS9 and LbCPF1 for creating KO mutations.

OsMADS26 Negatively Regulates Resistance to Pathogens and Drought Tolerance in Rice.

Functional analyses of MADS-box transcription factors in plants have unraveled their role in major developmental programs (e.g. flowering and floral organ identity) as well as stress-related developmental processes, such as abscission, fruit ripening, and senescence. Overexpression of the rice (Oryza sativa) MADS26 gene in rice has revealed a possible function related to stress response. Here, we show that OsMADS26-down-regulated plants exhibit enhanced resistance against two major rice pathogens: Magnaporthe oryzae and Xanthomonas oryzae. Despite this enhanced resistance to biotic stresses, OsMADS26-down-regulated plants also displayed enhanced tolerance to water deficit. These phenotypes were observed in both controlled and field conditions. Interestingly, alteration of OsMADS26 expression does not have a strong impact on plant development. Gene expression profiling revealed that a majority of genes misregulated in overexpresser and down-regulated OsMADS26 lines compared with control plants are associated to biotic or abiotic stress response. Altogether, our data indicate that OsMADS26 acts as an upstream regulator of stress-associated genes and thereby, a hub to modulate the response to various stresses in the rice plant.

Identification of CROWN ROOTLESS1-regulated genes in rice reveals specific and conserved elements of postembryonic root formation.

In monocotyledons, the root system is mostly composed of postembryonic shoot-borne roots called crown roots. In rice (Oryza sativa), auxin promotes crown root initiation via the LOB-domain transcription factor (LBD) transcription factor CROWN ROOTLESS1 (CRL1); however, the gene regulatory network downstream of CRL1 remains largely unknown. We tested CRL1 transcriptional activity in yeast and in planta, identified CRL1-regulated genes using an inducible gene expression system and a transcriptome analysis, and used in situ hybridization to demonstrate coexpression of a sample of CRL1-regulated genes with CRL1 in crown root primordia. We show that CRL1 positively regulates 277 genes, including key genes involved in meristem patterning (such as QUIESCENT-CENTER SPECIFIC HOMEOBOX; QHB), cell proliferation and hormone homeostasis. Many genes are homologous to Arabidopsis genes involved in lateral root formation, but about a quarter are rice-specific. Our study reveals that several genes acting downstream of LBD transcription factors controlling postembryonic root formation are conserved between monocots and dicots. It also provides evidence that specific genes are involved in the formation of shoot-derived roots in rice.

In-depth molecular and phenotypic characterization in a rice insertion line library facilitates gene identification through reverse and forward genetics approaches.

We report here the molecular and phenotypic features of a library of 31,562 insertion lines generated in the model japonica cultivar Nipponbare of rice (Oryza sativa L.), called Oryza Tag Line (OTL). Sixteen thousand eight hundred and fourteen T-DNA and 12,410 Tos17 discrete insertion sites have been characterized in these lines. We estimate that 8686 predicted gene intervals--i.e. one-fourth to one-fifth of the estimated rice nontransposable element gene complement--are interrupted by sequence-indexed T-DNA (6563 genes) and/or Tos17 (2755 genes) inserts. Six hundred and forty-three genes are interrupted by both T-DNA and Tos17 inserts. High quality of the sequence indexation of the T2 seed samples was ascertained by several approaches. Field evaluation under agronomic conditions of 27,832 OTL has revealed that 18.2% exhibit at least one morphophysiological alteration in the T1 progeny plants. Screening 10,000 lines for altered response to inoculation by the fungal pathogen Magnaporthe oryzae allowed to observe 71 lines (0.7%) developing spontaneous lesions simulating disease mutants and 43 lines (0.4%) exhibiting an enhanced disease resistance or susceptibility. We show here that at least 3.5% (four of 114) of these alterations are tagged by the mutagens. The presence of allelic series of sequence-indexed mutations in a gene among OTL that exhibit a convergent phenotype clearly increases the chance of establishing a linkage between alterations and inserts. This convergence approach is illustrated by the identification of the rice ortholog of AtPHO2, the disruption of which causes a lesion-mimic phenotype owing to an over-accumulation of phosphate, in nine lines bearing allelic insertions.

Transcript profiling of crown rootless1 mutant stem base reveals new elements associated with crown root development in rice.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Diversity of the Ty-1 copia retrotransposon Tos17 in rice (Oryza sativa L.) and the AA genome of the Oryza genus.

Retrotransposons are mobile genetic elements, ubiquitous in Eukaryotic genomes, which have proven to be major genetic tools in determining phylogeny and structuring genetic diversity, notably in plants. We investigate here the diversity of the Ty1-copia retrotransposon Tos17 in the cultivated rice of Asian origin (Oryza sativa L.) and related AA genome species of the Oryza genus, to contribute understanding of the complex evolutionary history in this group of species through that of the element in the lineages. In that aim, we used a combination of Southern hybridization with a reverse transcriptase (RT) probe and an adapter-PCR mediated amplification, which allowed the sequencing of the genomic regions flanking Tos17 insertions. This analysis was carried out in a collection of 47 A-genome Oryza species accessions and 202 accessions of a core collection of Oryza sativa L. representative of the diversity of the species. Our Southern hybridization results show that Tos17 is present in all the accessions of the A-genome Oryza species, except for the South American species O. glumaepatula and the African species O. glaberrima and O. breviligulata. In O. sativa, the number of putative copies of Tos17 per accession ranged from 1 to 11 and multivariate analysis based on presence/absence of putative copies yielded a varietal clustering which is consistent with the isozyme classification of rice. Adapter PCR amplification and sequencing of flanking regions of Tos17 insertions in A-genome species other than O. sativa, followed by anchoring on the Nipponbare genome sequence, revealed 13 insertion sites of Tos17 in the surveyed O. rufipogon and O. longistaminata accessions, including one shared by both species. In O. sativa, the same approach revealed 25 insertions in the 6 varietal groups. Four insertion sites located on chromosomes 1, 2, 10, and 11 were found orthologous in O. rufipogon and O. sativa. The chromosome 1 insertion was also shared between O. rufipogon and O. longistaminata. The presence of Tos17 at three insertion sites was confirmed by retrotransposon-based insertion polymorphism (RBIP) in a sample of O. sativa accessions. The first insertion, located on chromosome 3 was only found in two japonica accessions from the Bhutan region while the second insertion, located on chromosome 10 was specific to the varietal groups 1, 2, and 5. The third insertion located on chromosome 7 corresponds to the only insertion shown active in rice so far, notably in cv. Nipponbare, where it has been extensively used for insertion mutagenesis. This copy was only found in a few varieties of the japonica group 6 and in one group 5 accession. Taken together, these results confirm that Tos17 was probably present in the ancestor of A-genome species and that some copies of the element remained active in some Oryza lineages--notably in O. rufipogon and O. longistaminata--as well as in the indica and japonica O. sativa L. lineages.

Large-scale characterization of Tos17 insertion sites in a rice T-DNA mutant library.

We characterized the insertion sites of newly transposed copies of the tissue-culture-induced ty1-copia retrotransposon Tos17 in the Oryza Tag Line (OTL) T-DNA mutant library of rice cv. Nipponbare. While Nipponbare contains two native copies of Tos17 the number of additional copies, deduced from Southern blot analyses in a subset of 384 T-DNA lines and using a reverse transcriptase probe specific to the element, ranged from 1 to 8 and averaged 3.37. These copies were shown to be stably inherited and to segregate independently in the progenies of insertion lines. We took advantage of the absence of EcoRV restriction sites in the immediate vicinity of the 3' LTR of the native copies of Tos17 in the genome sequence of cv. Nipponbare, thereby preventing amplification of corresponding PCR fragments, to efficiently and selectively amplify and sequence flanking regions of newly transposed Tos17 inserts. From 25,286 T-DNA plants, we recovered 19,252 PCR products (76.1%), which were sequenced yielding 14,513 FSTs anchored on the rice pseudomolecules. Following elimination of redundant sequences due to the presence of T-DNA plants deriving from the same cell lineage, these FSTs corresponded to 11,689 unique insertion sites. These unique insertions exhibited higher densities in subtelomeric regions of the chromosomes and hot spots for integration, following a distribution that remarkably paralleled that of Tos17 sites in the National Institute for Agrobiological Sciences (NIAS) library. The insertion sites were mostly found in genic regions (77.5%) and preferably in coding sequences (68.8%) compared to unique T-DNA insertion sites in the same materials (49.1% and 28.3%, respectively). Predicted non- transposable element (TE) genes prone to a high frequency of Tos17 integration (i.e. from 5 to 121 inserts) in the OTL T-DNA collection were generally found to be also hot spots for integration in the NIAS library. The 9,060 Tos17 inserts inserted into non TE genes were found to disrupt a total of 2,773 genes with an average of 3.27 inserts per gene, similar to that in the NIAS library (3.28 inserts per gene on average) whereas the 4,472 T-DNA inserted into genes in the same materials disrupted a total of 3,911 genes (1.14 inserts per gene on average). Interestingly, genes disrupted by both Tos17 and T-DNA inserts in the library represented only 14.9% and 10.6% of the complement of genes interrupted by Tos17 and T-DNA inserts respectively while 52.1% of the genes tagged by Tos17 inserts in the OTL library were found to be tagged also in the NIAS Tos17 library. We concluded that the first advantage in characterizing Tos17 inserts in a rice T-DNA collection lies in a complementary tagging of novel genes and secondarily in finding other alleles in a same genetic background, thereby greatly enhancing the library genome coverage and its overall value for implementing forward and reverse genetics strategies.

High throughput T-DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics.

A library of 29,482 T-DNA enhancer trap lines has been generated in rice cv. Nipponbare. The regions flanking the T-DNA left border from the first 12,707 primary transformants were systematically isolated by adapter anchor PCR and sequenced. A survey of the 7480 genomic sequences larger than 30 bp (average length 250 bp), representing 56.4% of the total readable sequences and matching the rice bacterial artificial chromosome/phage artificial chromosome (BAC/PAC) sequences assembled in pseudomolecules allowed the assigning of 6645 (88.8%) T-DNA insertion sites to at least one position in the rice genome of cv. Nipponbare. T-DNA insertions appear to be rather randomly distributed over the 12 rice chromosomes, with a slightly higher insertion frequency in chromosomes 1, 2, 3 and 6. The distribution of 723 independent T-DNA insertions along the chromosome 1 pseudomolecule did not differ significantly from that of the predicted coding sequences in exhibiting a lower insertion density around the centromere region and a higher density in the subtelomeric regions where the gene density is higher. Further establishment of density graphs of T-DNA inserts along the recently released 12 rice pseudomolecules confirmed this non-uniform chromosome distribution. T-DNA appeared less prone to hot spots and cold spots of integration when compared with those revealed by a concurrent assignment of the Tos17 retrotransposon flanking sequences deposited in the National Center for Biotechnology Information (NCBI). T-DNA inserts rarely integrated into repetitive sequences. Based on the predicted gene annotation of chromosome 1, preferential insertion within the first 250 bp from the putative ATG start codon has been observed. Using 4 kb of sequences surrounding the insertion points, 62% of the sequences showed significant similarity to gene encoding known proteins (E-value < 1.00 e(-05)). To illustrate the in silico reverse genetic approach, identification of 83 T-DNA insertions within genes coding for transcription factors (TF) is presented. Based both on the estimated number of members of several large TF gene families (e.g. Myb, WRKY, HD-ZIP, Zinc-finger) and on the frequency of insertions in chromosome 1 predicted genes, we could extrapolate that 7-10% of the rice gene complement is already tagged by T-DNA insertion in the 6116 independent transformant population. This large resource is of high significance while assisting studies unravelling gene function in rice and cereals, notably through in silico reverse genetics.

Albicidin pathotoxin produced by Xanthomonas albilineans is encoded by three large PKS and NRPS genes present in a gene cluster also containing several putative modifying, regulatory, and resistance genes.

Xanthomonas albilineans, which causes leaf scald disease of sugarcane, produces a highly potent pathotoxin called albicidin. We report here sequencing and homology analysis of the major gene cluster, XALB1 (55,839 bp), and a second, smaller region, XALB2 (2,986 bp), involved in albicidin biosynthesis. XALB1 contains 20 open reading frames, including i) three large genes with a modular architecture characteristic of polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and ii) several putative modifying, regulatory, and resistance genes. Sequencing and complementation studies of six albicidin-defective mutants enabled us to confirm the involvement of the three PKS and NRPS genes encoded by XALB1 in albicidin production. XALB2 contains only one gene that is required for post-translational activation of PKS and NRPS enzymes, confirming the involvement of these enzymes in albicidin biosynthesis. In silico analysis of these three PKS or NRPS enzymes allowed us to propose a model for the albicidin backbone assembly and to gain insight into the structural features of this pathotoxin. This is the first description of a complete mixed PKS-NRPS gene cluster for toxin production in the genus Xanthomonas.