CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.

Cytoplasmic male sterility (CMS) and nucleus-controlled fertility restoration are widespread plant reproductive features that provide useful tools to exploit heterosis in crops. However, the molecular mechanism underlying this kind of cytoplasmicnuclear interaction remains unclear. Here, we show in rice (Oryza sativa) with Boro II cytoplasm that an abnormal mitochondrial open reading frame, orf79, is cotranscribed with a duplicated atp6 (B-atp6) gene and encodes a cytotoxic peptide. Expression of orf79 in CMS lines and transgenic rice plants caused gametophytic male sterility. Immunoblot analysis showed that the ORF79 protein accumulates specifically in microspores. Two fertility restorer genes, Rf1a and Rf1b, were identified at the classical locus Rf-1 as members of a multigene cluster that encode pentatricopeptide repeat proteins. RF1A and RF1B are both targeted to mitochondria and can restore male fertility by blocking ORF79 production via endonucleolytic cleavage (RF1A) or degradation (RF1B) of dicistronic B-atp6/orf79 mRNA. In the presence of both restorers, RF1A was epistatic over RF1B in the mRNA processing. We have also shown that RF1A plays an additional role in promoting the editing of atp6 mRNAs, independent of its cleavage function.

Plant cytoplasmic male sterility (CMS) results from incompatibilities between the organellar and nuclear genomes and prevents self pollination, enabling hybrid crop breeding to increase yields. The Wild Abortive CMS (CMS-WA) has been exploited in the majority of 'three-line' hybrid rice production since the 1970s, but the molecular basis of this trait remains unknown. Here we report that a new mitochondrial gene, WA352, which originated recently in wild rice, confers CMS-WA because the protein it encodes interacts with the nuclear-encoded mitochondrial protein COX11. In CMS-WA lines, WA352 accumulates preferentially in the anther tapetum, thereby inhibiting COX11 function in peroxide metabolism and triggering premature tapetal programmed cell death and consequent pollen abortion. WA352-induced sterility can be suppressed by two restorer-of-fertility (Rf) genes, suggesting the existence of different mechanisms to counteract deleterious cytoplasmic factors. Thus, CMS-related cytoplasmic-nuclear incompatibility is driven by a detrimental interaction between a newly evolved mitochondrial gene and a conserved, essential nuclear gene.

Sterility is common in hybrids between divergent populations, such as the indica and japonica subspecies of Asian cultivated rice (Oryza sativa). Although multiple loci for plant hybrid sterility have been identified, it remains unknown how alleles of the loci interact at the molecular level. Here we show that a locus for indicajaponica hybrid male sterility, Sa, comprises two adjacent genes, SaM and SaF, encoding a small ubiquitin-like modifier E3 ligase-like protein and an F-box protein, respectively. Most indica cultivars contain a haplotype SaM ؉ SaF ؉ , whereas all japonica cultivars have SaM ؊ SaF ؊ that diverged by nucleotide variations in wild rice. Male semi-sterility in this heterozygous complex locus is caused by abortion of pollen carrying SaM ؊ . This allele-specific gamete elimination results from a selective interaction of SaF ؉ with SaM ؊ , a truncated protein, but not with SaM ؉ because of the presence of an inhibitory domain, although SaM ؉ is required for this male sterility. Lack of any one of the three alleles in recombinant plants does not produce male sterility. We propose a two-gene/threecomponent interaction model for this hybrid male sterility system. The findings have implications for overcoming male sterility in inter-subspecific hybrid rice breeding.allelic interaction ͉ gamete selection ͉ hybrid sterility ͉ reproductive barrier ͉ two-gene/three-component model

Anthocyanins have high antioxidant activities, and engineering of anthocyanin biosynthesis in staple crops, such as rice (Oryza sativa L.), could provide health-promoting foods for improving human health. However, engineering metabolic pathways for biofortification remains difficult, and previous attempts to engineer anthocyanin production in rice endosperm failed because of the sophisticated genetic regulatory network of its biosynthetic pathway. In this study, we developed a high-efficiency vector system for transgene stacking and used it to engineer anthocyanin biosynthesis in rice endosperm. We made a construct containing eight anthocyanin-related genes (two regulatory genes from maize and six structural genes from Coleus) driven by the endosperm-specific promoters,plus a selectable marker and a gene for marker excision. Transformation of rice with this construct generated a novel biofortified germplasm "Purple Endosperm Rice" (called "Zijingmi" in Chinese), which has high anthocyanin contents and antioxidant activity in the endosperm. This anthocyanin production results from expression of the transgenes and the resulting activation (or enhancement) of expression of 13 endogenous anthocyanin biosynthesis genes that are silenced or expressed at low levels in wild-type rice endosperm. This study provides an efficient, versatile toolkit for transgene stacking and demonstrates its use for successful engineering of a sophisticated biological pathway, suggesting the potential utility of this toolkit for synthetic biology and improvement of agronomic traits in plants.

Ahmadikhah, A., and karlov, G.I. (2006). Molecular mapping of the fertility-restoration gene Rf4 for WA-cytoplasmic male sterility in rice. Plant Breeding. 125, 363-367.

No abstract

In the absence of pathogen attack, organisms usually suppress immune responses to reduce the negative effects of disease resistance. Monoubiquitination of histone variants at specific gene loci is crucial for gene expression, but its involvement in the regulation of plant immunity remains unclear. Here, we show that a rice SWI/SNF2 ATPase gene BRHIS1 is downregulated in response to the rice blast fungal pathogen or to the defensepriming-inducing compound BIT (1,2-benzisothiazol-3(2h)-one,1, 1-dioxide). The BRHIS1-containing complex represses the expression of some disease defense-related genes, including the pathogenesis-related gene OsPBZc and the leucine-rich-repeat (LRR) receptor-like protein kinase gene OsSIRK1. This is achieved through BRHIS1 recruitment to the promoter regions of target genes through specific interaction with monoubiquitinated histone variants H2B.7 and H2A.Xa/H2A.Xb/H2A.3, in the absence of pathogen attack or BIT treatment. Our results show that rice disease defense genes are initially organized in an expressionready state by specific monoubiquitination of H2A and H2B variants deposited on their promoter regions, but are kept suppressed by the BRHIS1 complex, facilitating the prompt initiation of innate immune responses in response to infection through the stringent regulation of BRHIS1.