Clonal reproduction by seed of a cultivated hybrid plant: a new perspective for small-scale rice growers.

Transferring an asexual mode of reproduction by seeds (apomixis) to cultivated plants would enable clonal reproduction of heterozygous genotypes such as F1 hybrids with hybrid vigor (heterosis), facilitating their access and multiplication by small-scale growers. Although sources of apomixis and the genetic loci controlling its constituent elements have been identified in wild species, their transfer by crossing to cultivated species has so far been unsuccessful. Here, we have introduced synthetic apomixis in hybrid rice to produce a high (95-100%) frequency of clonal seeds, via the inactivation of three meiotic genes-resulting in unreduced, non-recombined gametes-and the addition of an egg cell parthenogenesis trigger. The genotype and phenotype, including grain quality, of the F1 hybrid are reproduced identically in the clonal apomictic progenies. These results make synthetic apomixis compatible with future use in agriculture.

Le transfert d'un mode de reproduction clonale asexuée par grain (apomixie) aux plantes cultivées permettrait de reproduire de façon génétiquement identique des génotypes hétérozygotes comme ceux des hybrides F1 dotés d'une vigueur hybride (heterosis), facilitant ainsi leur accès et leur multiplication par les petits cultivateurs. Bien que des sources d'apomixie et les loci génétiques contrôlant ses éléments constitutifs aient été identifiés chez les espèces sauvages, leur transfert par croisement aux espèces cultivées a jusqu'à présent été infructueux. Ici, nous avons introduit chez un riz hybride une apomixie synthétique produisant une haute fréquence de grains clonaux (95­100%), via l'inactivation de trois gènes méiotiques ­ permettant d'obtenir des gamètes non réduits et non recombinés ­ et l'apport d'un déclencheur de la parthénogenèse. Le génotype et le phénotype, incluant la qualité de grain, de l'hybride F1 sont reproduits à l'identique dans les descendances apomictiques clonales. Ces résultats rendent compatible l'apomixie synthétique avec une future utilisation en agriculture.

High-frequency synthetic apomixis in hybrid rice.

Introducing asexual reproduction through seeds - apomixis - into crop species could revolutionize agriculture by allowing F1 hybrids with enhanced yield and stability to be clonally propagated. Engineering synthetic apomixis has proven feasible in inbred rice through the inactivation of three genes (MiMe), which results in the conversion of meiosis into mitosis in a line ectopically expressing the BABYBOOM1 (BBM1) parthenogenetic trigger in egg cells. However, only 10-30% of the seeds are clonal. Here, we show that synthetic apomixis can be achieved in an F1 hybrid of rice by inducing MiMe mutations and egg cell expression of BBM1 in a single step. We generate hybrid plants that produce more than 95% of clonal seeds across multiple generations. Clonal apomictic plants maintain the phenotype of the F1 hybrid along successive generations. Our results demonstrate that there is no barrier to almost fully penetrant synthetic apomixis in an important crop species, rendering it compatible with use in agriculture.

Manipulation of Meiotic Recombination to Hasten Crop Improvement.

Reciprocal (cross-overs = COs) and non-reciprocal (gene conversion) DNA exchanges between the parental chromosomes (the homologs) during meiotic recombination are, together with mutation, the drivers for the evolution and adaptation of species. In plant breeding, recombination combines alleles from genetically diverse accessions to generate new haplotypes on which selection can act. In recent years, a spectacular progress has been accomplished in the understanding of the mechanisms underlying meiotic recombination in both model and crop plants as well as in the modulation of meiotic recombination using different strategies. The latter includes the stimulation and redistribution of COs by either modifying environmental conditions (e.g., T°), harnessing particular genomic situations (e.g., triploidy in Brassicaceae), or inactivating/over-expressing meiotic genes, notably some involved in the DNA double-strand break (DSB) repair pathways. These tools could be particularly useful for shuffling diversity in pre-breeding generations. Furthermore, thanks to the site-specific properties of genome editing technologies the targeting of meiotic recombination at specific chromosomal regions nowadays appears an attainable goal. Directing COs at desired chromosomal positions would allow breaking linkage situations existing between favorable and unfavorable alleles, the so-called linkage drag, and accelerate genetic gain. This review surveys the recent achievements in the manipulation of meiotic recombination in plants that could be integrated into breeding schemes to meet the challenges of deploying crops that are more resilient to climate instability, resistant to pathogens and pests, and sparing in their input requirements.

Assessment of the roles of SPO11-2 and SPO11-4 in meiosis in rice using CRISPR/Cas9 mutagenesis.

In Arabidopsis, chromosomal double-strand breaks at meiosis are presumably catalyzed by two distinct SPO11 transesterases, AtSPO11-1 and AtSPO11-2, together with M-TOPVIB. To clarify the roles of the SPO11 paralogs in rice, we used CRISPR/Cas9 mutagenesis to produce null biallelic mutants in OsSPO11-1, OsSPO11-2, and OsSPO11-4. Similar to Osspo11-1, biallelic mutations in the first exon of OsSPO11-2 led to complete panicle sterility. Conversely, all Osspo11-4 biallelic mutants were fertile. To generate segregating Osspo11-2 mutant lines, we developed a strategy based on dual intron targeting. Similar to Osspo11-1, the pollen mother cells of Osspo11-2 progeny plants showed an absence of bivalent formation at metaphase I, aberrant segregation of homologous chromosomes, and formation of non-viable tetrads. In contrast, the chromosome behavior in Osspo11-4 male meiocytes was indistinguishable from that in the wild type. While similar numbers of OsDMC1 foci were revealed by immunostaining in wild-type and Osspo11-4 prophase pollen mother cells (114 and 101, respectively), a surprisingly high number (85) of foci was observed in the sterile Osspo11-2 mutant, indicative of a divergent function between OsSPO11-1 and OsSPO11-2. This study demonstrates that whereas OsSPO11-1 and OsSPO11-2 are the likely orthologs of AtSPO11-1 and AtSPO11-2, OsSPO11-4 has no major role in wild-type rice meiosis.

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.

Sub-cellular markers highlight intracellular dynamics of membrane proteins in response to abiotic treatments in rice.

BACKGROUND: Cell biology approach using membrane protein markers tagged with fluorescent proteins highlights the dynamic behaviour of plant cell membranes, not only in the standard but also in changing environmental conditions. In the past, this strategy has been extensively developed in plant models such as Arabidopsis. RESULTS: Here, we generated a set of transgenic lines expressing membrane protein markers to extend this approach to rice, one of the most cultivated crop in the world and an emerging plant model. Lines expressing individually eight membrane protein markers including five aquaporins (OsPIP1;1, OsPIP2;4, OsPIP2;5, OsTIP1;1, OsTIP2;2) and three endosomal trafficking proteins (OsRab5a, OsGAP1, OsSCAMP1) were obtained. Importantly, we challenged in roots the aquaporin-expressing transgenic lines upon salt and osmotic stress and uncovered a highly dynamic behaviour of cell membrane. CONCLUSION: We have uncovered the relocalization and dynamics of plasma membrane aquaporins upon salt and osmotic stresses in rice. Importantly, our data support a model where relocalization of OsPIPs is concomitant with their high cycling dynamics.

Tolerance to mild salinity stress in japonica rice: A genome-wide association mapping study highlights calcium signaling and metabolism genes.

Salinity tolerance is an important quality for European rice grown in river deltas. We evaluated the salinity tolerance of a panel of 235 temperate japonica rice accessions genotyped with 30,000 SNP markers. The panel was exposed to mild salt stress (50 mM NaCl; conductivity of 6 dS m-1) at the seedling stage. Eight different root and shoot growth parameters were measured for both the control and stressed treatments. The Na+ and K+ mass fractions of the stressed plants were measured using atomic absorption spectroscopy. The salt treatment affected plant growth, particularly the shoot parameters. The panel showed a wide range of Na+/K+ ratio and the temperate accessions were distributed over an increasing axis, from the most resistant to the most susceptible checks. We conducted a genome-wide association study on indices of stress response and ion mass fractions in the leaves using a classical mixed model controlling structure and kinship. A total of 27 QTLs validated by sub-sampling were identified. For indices of stress responses, we also used another model that focused on marker × treatment interactions and detected 50 QTLs, three of which were also identified using the classical method. We compared the positions of the significant QTLs to those of approximately 300 genes that play a role in rice salt tolerance. The positions of several QTLs were close to those of genes involved in calcium signaling and metabolism, while other QTLs were close to those of kinases. These results reveal the salinity tolerance of accessions with a temperate japonica background. Although the detected QTLs must be confirmed by other approaches, the number of associations linked to candidate genes involved in calcium-mediated ion homeostasis highlights pathways to explore in priority to understand the salinity tolerance of temperate rice.

Production of low-Cs+ rice plants by inactivation of the K+ transporter OsHAK1 with the CRISPR-Cas system.

The occurrence of radiocesium in food has raised sharp health concerns after nuclear accidents. Despite being present at low concentrations in contaminated soils (below µm), cesium (Cs+ ) can be taken up by crops and transported to their edible parts. This plant capacity to take up Cs+ from low concentrations has notably affected the production of rice (Oryza sativa L.) in Japan after the nuclear accident at Fukushima in 2011. Several strategies have been put into practice to reduce Cs+ content in this crop species such as contaminated soil removal or adaptation of agricultural practices, including dedicated fertilizer management, with limited impact or pernicious side-effects. Conversely, the development of biotechnological approaches aimed at reducing Cs+ accumulation in rice remain challenging. Here, we show that inactivation of the Cs+ -permeable K+ transporter OsHAK1 with the CRISPR-Cas system dramatically reduced Cs+ uptake by rice plants. Cs+ uptake in rice roots and in transformed yeast cells that expressed OsHAK1 displayed very similar kinetics parameters. In rice, Cs+ uptake is dependent on two functional properties of OsHAK1: (i) a poor capacity of this system to discriminate between Cs+ and K+ ; and (ii) a high capacity to transport Cs+ from very low external concentrations that is likely to involve an active transport mechanism. In an experiment with a Fukushima soil highly contaminated with 137 Cs+ , plants lacking OsHAK1 function displayed strikingly reduced levels of 137 Cs+ in roots and shoots. These results open stimulating perspectives to smartly produce safe food in regions contaminated by nuclear accidents.

Turning rice meiosis into mitosis.

Introduction of clonal reproduction through seeds (apomixis) in crops has the potential to revolutionize agriculture by allowing self-propagation of any elite variety, in particular F1 hybrids. In the sexual model plant Arabidopsis thaliana synthetic clonal reproduction through seeds can be artificially implemented by (i) combining three mutations to turn meiosis into mitosis (MiMe) and (ii) crossing the obtained clonal gametes with a line expressing modified CENH3 and whose genome is eliminated in the zygote. Here we show that additional combinations of mutations can turn Arabidopsis meiosis into mitosis and that a combination of three mutations in rice (Oryza sativa) efficiently turns meiosis into mitosis, leading to the production of male and female clonal diploid gametes in this major crop. Successful implementation of the MiMe technology in the phylogenetically distant eudicot Arabidopsis and monocot rice opens doors for its application to any flowering plant and paves the way for introducing apomixis in crop species.

Functional analysis of the durum wheat gene TdPIP2;1 and its promoter region in response to abiotic stress in rice.

In a previous work, we demonstrated that expression of TdPIP2;1 in Xenopus oocytes resulted in an increase in Pf compared to water injected oocytes. Phenotypic analyses of transgenic tobacco plants expressing TdPIP2;1 generated a tolerance phenotype towards drought and salinity stresses. To elucidate its stress tolerance mechanism at the transcriptional level, we isolated and characterized the promoter region of the TdPIP2;1 gene. A 1060-bp genomic fragment upstream of the TdPIP2;1 translated sequence has been isolated, cloned, and designated as the proTdPIP2;1 promoter. Sequence analysis of proTdPIP2;1 revealed the presence of cis regulatory elements which could be required for abiotic stress responsiveness, for tissue-specific and vascular expression. The proTdPIP2;1 promoter was fused to the ß-glucuronidase (gusA) gene and the resulting construct was transferred into rice (cv. Nipponbare). Histochemical analysis of proTdPIP2;1::Gus in rice plants revealed that the GUS activity was observed in leaves, stems and roots of stably transformed rice T3 plants. Histological sections prepared revealed accumulation of GUS products in phloem, xylem and in some cells adjacent to xylem. The transcripts were up-regulated by dehydration. Transgenic rice plants overexpressing proTdPIP2;1 in fusion with TdPIP2;1, showed enhanced drought tolerance, while wild type plants were more sensitive and exhibited symptoms of wilting and chlorosis. These findings suggest that expression of the TdPIP2;1 gene regulated by its own promoter achieves enhanced drought tolerance in rice.