Rice Callus Research Articles

The effect of plant growth regulators, carbon sources and gelling agent on the callus regeneration of Malaysian rice MR219 (Oryza sativa L.)

The development of a highly efficient tissue culture system for indica rice is crucial to speed up the implementation of gene editing in breeding programmes as well as to explore the roles of indica genes. However, because indica rice types are recalcitrant to in vitro response, there are only a few reports on the successful transformation process on indica rice due to recalcitrant response to in vitro response. The present study attempted to identify the effects of plant growth regulator (PGR), carbon sources and different concentrations of gelling agent on embryogenic callus induction and rice regeneration of Oryza sativa var MR219. The PGR used were 1 or 2 mg/L 6-benzylaminopurine (BAP) and 1-Naphthaleneacetic acid (NAA) and the carbon sources were 30 g/L sucrose and maltose. Three different gelling agent concentrations (3 mg/L, 6 mg/L or 9 mg/L) were evaluated. The highest frequency of friable callus induction (87%) was observed in callus induction media (CI) containing Murashige and Skoong media (MS) supplemented with 3 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.3 mg/L kinetin. Histological studies of the calli showed the presence of embryogenic calli with small intercellular spaces compared to non-embryogenic areas which developed on the hormone MS media. The highest regeneration frequency (50%) was achieved when friable calli were cultured on MS medium containing 3% sucrose, 1 mg/L BAP, and 1 mg/L NAA with 9% phytagel. The callus grown on sucrose media showed the highest number (66.13%) of albino plantlets, while the number of albino plantlets on maltose media was smaller (20%). In conclusion, this optimised mature embryo-derived callus of MR219 rice is responsive to regeneration and could be amenable to genetic transformation studies.

The sonication-assisted whisker method enables CRISPR-Cas9 ribonucleoprotein delivery to induce genome editing in rice

CRISPR/Cas9-based genome editing represents an unprecedented potential for plant breeding. Unlike animal cells, plant cells contain a rigid cell wall, genome editing tool delivery into plant cells is thus challenging. In particular, the delivery of the Cas9-gRNA ribonucleoprotein (RNP) into plant cells is desired since the transgene insertion into the genome should be avoided for industrial applications in plants. In this study, we present a novel RNP delivery approach in rice. We applied the sonication-assisted whisker method, conventionally developed for DNA delivery in plants, for RNP delivery in rice. Combined with marker gene delivery, we successfully isolated OsLCYβ genome-edited lines generated by RNPs. The calli and regenerated shoot of the OsLCYβ mutant showed abnormal carotenoid accumulation. In addition, we also detected, although at a low frequency, genome editing events in rice calli cells by RNP delivery using the sonication-assisted whisker method without any additional. Therefore, the sonication-assisted whisker method could be an attractive way to create RNP-based genome-edited lines in plants.

The Synergistic Effects of Environmental and Genetic Factors on the Regulation of Anthocyanin Accumulation in Plant Tissues.

Anthocyanin accumulation is responsible for the coloration of apple fruit, and their accumulation depends on the expression of anthocyanin biosynthesis-related genes. Light is an environmental stimulus that induces fruit color by regulating genes involved in the anthocyanin biosynthesis pathway. In this study, the roles of light and genetic factors on fruit coloration and anthocyanin accumulation in apple fruit were investigated. Three genes in the anthocyanin biosynthesis pathway, MdCHS, MdANS, and MdUFGT1, were synthesized and cloned into a viral-based expression vector system for transient expression in 'Ruby S' apple fruits. Apple fruits were agroinfiltrated with expression vectors harboring MdCHS, MdANS, and MdUFGT1. Agroinfiltrated apple fruits were then either kept in the dark (bagged fruits) or exposed to light (exposed fruits). The agroinfiltrated fruits showed significantly different coloration patterns, transcript expression levels, and anthocyanin accumulation compared to the control fruits. Moreover, these parameters were higher in exposed fruits than in bagged fruits. For stable expression, MdCHS was introduced into a binary vector under the control of the rice α-amylase 3D (RAmy3D) promoter. The ectopic overexpression of MdCHS in transgenic rice calli showed a high accumulation of anthocyanin content. Taken together, our findings suggest that light, together with the overexpression of anthocyanin biosynthesis genes, induced the coloration and accumulation of anthocyanin content in apple fruits by upregulating the expression of the genes involved in the anthocyanin biosynthesis pathway.

The Influence of Plant Growth Regulators on Callus Induction in Mature Rice Embryos (Oryza Sativa L) Amber Variety

Several nations across the globe depend profoundly on rice as the main food source. Several research works have been undertaken to improve rice yield and breeding, specifically concerning plant tissue culture. This paper aims to contrast the impact of several growth regulator levels on rice callus induction based on mature embryo explants. Over a four-week duration, mature rice plant embryos (Amber variant) were planted to produce calluses using the Skoog (MS) and Murashige supplements at different concentration levels, i.e., 2.0, 2.5, and 3.0 mg/L of 2,4-D and 0.2 and 0.3 mg/L of BA. The outcomes indicated that 3 mg/L 2,4-D and 0.2 mg/L BA levels led to optimal induction and fresh weight concerning callus. Further studies are required to enhance the media environment for potent in vitro callus induction to augment biotechnology endeavours.

Direct observation of common cryoprotectant permeation into rice callus by CARS microscopy

Direct observation of common cryoprotectant permeation into rice callus by CARS microscopy

Screening Nos-bar gene of Transgenic Rice Callus (Oryza Sativa L.) Containing Antihypertensive Gene Encoding Gg-7pAH

Peptides derived from food protein have the potential to become antihypertensive agents with relatively few adverse side effects. The Gg-7pAH gene encoding antihypertensive is a gene containing the Cys-Met-Try-Leu-Ala-Ser-Gly (CMYLASG) heptapeptide, angiotensin-converting enzyme (ACE) inhibitor peptide from Melinjo (Gnetum gnemon L) seeds. This study aimed to investigate the expression of the tandem repeat heptapeptide (10x peptides, Gg7-pAH) and Nos-bar genes through callus resistance to phosphinothricin (PPT) in 4 different phases of callus: co-cultivation, elimination, screening, and regeneration. The Gg7-pAH gene was constructed in the pGreen II 0229 vector containing the Nos-bar gene and  Gg7-pAH gene under the control of the Glu4 promoter and transformed into rice callus using an Agrobacterium tumefacient vector. PPT was used in the screening at precise concentrations of 0, 20, 50, and 100 ppm. The screening efficiency was then evaluated by calculating the putative callus. As a result, 37 putative callus were developed with a concentration of 0 ppm PPT, 36 putative callus with a concentration of 20 ppm PPT, and none at 50 and 100 ppm PPT. The Gg-7pAH gene was identified by PCR analysis in putatively transgenic rice callus. According to PCR data, a positive callus with an antihypertensive gene insertion was detected at four phases: media, co-cultivation, elimination, screening, and regeneration. These findings suggest that the recombinant Gg-7pAH gene, which is transformed into rice plants to produce rice plants that can treat hypertension, has some potential.

Biopotentials of Collagen Scaffold Impregnated with Plant-Cell-Derived Epidermal Growth Factor in Defective Bone Healing.

The combination of scaffolds with recombinant human epidermal growth factor (rhEGF) protein can enhance defective bone healing via synergistic activation to stimulate cellular growth, differentiation, and survival. We examined the biopotentials of an rhEGF-loaded absorbable collagen scaffold (ACS) using a mouse model of calvarial defects, in which the rhEGF was produced from a plant cell suspension culture system because of several systemic advantages. Here, we showed a successful and large-scale production of plant-cell-derived rhEGF protein (p-rhEGF) by introducing an expression vector that cloned with its cDNA under the control of rice α-amylase 3D promoter into rice calli (Oryza sativa L. cv. Dongjin). Implantation with p-rhEGF (5 μg)-loaded ACSs into critical-sized calvarial defects enhanced new bone formation and the expression of osteoblast-specific markers in the defected regions greater than implantation with ACSs alone did. The potency of p-rhEGF-induced bone healing was comparable with that of Escherichia coli-derived rhEGF protein. The exogenous addition of p-rhEGF increased the proliferation of human periodontal ligament cells and augmented the induction of interleukin 8, bone morphogenetic protein 2, and vascular endothelial growth factor in the cells. Collectively, this study demonstrates the successful and convenient production of p-rhEGF, as well as its potency to enhance ACS-mediated bone regeneration by activating cellular responses that are required for wound healing.

Overexpression of type II rice metacaspase, OsMC4, increases endoplasmic reticulum stress tolerance in transgenic rice calli

The endoplasmic reticulum (ER) is an organelle responsible for regulating protein synthesis in plants. High salinity can lead to the accumulation of misfolded proteins, resulting in an ER stress response mechanism known as the unfolded protein response (UPR). Failure of the UPR to reverse the effect of protein misfolding will activate programmed cell death (PCD). Metacaspase genes are known regulate PCD in plants. This study aims to provide a comprehensive analysis of the expression patterns of type II rice metacaspase (OsMC) genes in response to ER and salinity stress in rice leaf. Among five type II metacaspases in the rice genome, OsMC4, OsMC5, and OsMC8 expressions were found to be upregulated during treatment with tunicamycin (ER stress) and sodium chloride, NaCl (salinity stress). A construct of taqRFP::OsMC4, controlled by the CaMV35S promoter, was generated and transformed into rice calli. The transgenic rice calli overexpressing taqRFP::OsMC4 demonstrated significant changes in the expression of the ER stress-marker genes, protein disulfide isomerase (OsPDI), and binding immunoglobulin protein (OsBiP). The results from this analysis provide preliminary evidence that at least one of the type II metacaspases, OsMC4, is be able to reduce ER and salinity stresses in rice. Further functional analysis of OsMC genes in ER and salinity stress tolerance could be carried out in transgenic rice overexpressing OsMCs in the future to improve stress tolerance. Simple summaryOryza sativa, commonly known as rice, is one of the most consumed crops in the world. In response to multiple biotic and abiotic factors, a series of endoplasmic reticulum (ER) stress response regulators are activated. There is evidence that high salinity triggers ER stress in plants. This study aims to determine the level of gene expression among type II metacaspases in rice in response to ER and salinity stress and to assess how they may be linked to PCD in rice calli. Three metacaspase genes, OsMC4, OsMC5, and OsMC8, have been observed to have significant expression post-treatment with tunicamycin in rice leaf. Overexpression of taqRFP::OsMC4 in rice calli significantly reduces the expression level of the stress markers, OsBiP and OsPDI, indicating that the stress level is relatively lower in the transgenic calli compared to the wild-type calli. Therefore, overexpression of taqRFP::OsMC4 in rice may increase rice tolerance towards ER and salinity stress. These expression analyses of the OsMCs family provide valuable information for further functional studies on the biological roles of OsMCs in ER and salinity stress responses.

Editing efficiencies with Cas9 orthologs, Cas12a endonucleases, and temperature in rice.

The advent of CRISPR-Cas technology has made it the genome editing tool of choice in all kingdoms of life, including plants, which can have large, highly duplicated genomes. As a result, finding adequate target sequences that meet the specificities of a given Cas nuclease on any gene of interest remains challenging in many cases. To assess target site flexibility, we tested five different Cas9/Cas12a endonucleases (SpCas9, SaCas9, St1Cas9, Mb3Cas12a, and AsCas12a) in embryogenic rice calli from Taipei 309 at 37°C (optimal temperature for most Cas9/Cas12a proteins) and 27°C (optimal temperature for tissue culture) and measured their editing rates under regular tissue culture conditions using Illumina sequencing. StCas9 and AsCas12 were not functional as tested, regardless of the temperature used. SpCas9 was the most efficient endonuclease at either temperature, regardless of whether monoallelic or biallelic edits were considered. Mb3Cas12a at 37°C was the next most efficient endonuclease. Monoallelic edits prevailed for both SaCas9 and Mb3Cas12a at 27°C, but biallelic edits prevailed at 37°C. Overall, the use of other Cas9 orthologs, the use of Cas12a endonucleases, and the optimal temperature can expand the range of targetable sequences.

Genome editing in rice mediated by miniature size Cas nuclease SpCas12f.

Cas9 derived from Streptococcus pyogenes (SpCas9) is used widely in genome editing using the CRISPR-Cas system due to its high activity, but is a relatively large molecule (1,368 amino acid (a.a.) residues). Recently, targeted mutagenesis in human cells and maize using Cas12f derived from Syntrophomonas palmitatica (SpCas12f)-a very small Cas of 497 a.a, which is a more suitable size for virus vectors-was reported. However, there are no reports of genome editing using SpCas12f in crops other than maize. In this study, we applied SpCas12f to genome editing in rice-one of the most important staple crops in the world. An expression vector encoding rice codon-optimized SpCas12f and sgRNA for OsTubulin as a target was introduced into rice calli by Agrobacterium-mediated transformation. Molecular analysis of SpCas12f-transformed calli showed that mutations were introduced successfully into the target region. Detailed analysis by amplicon sequencing revealed estimated mutation frequencies (a ratio of the number of mutated calli to that of SpCas12f-transformed calli) of 28.8% and 55.6% in two targets. Most mutation patterns were deletions, but base substitutions and insertions were also confirmed at low frequency. Moreover, off-target mutations by SpCas12f were not found. Furthermore, mutant plants were regenerated successfully from the mutated calli. It was confirmed that the mutations in the regenerated plants were inherited to the next-generation. In the previous report in maize, mutations were introduced by treatment with heat shock at 45°C for 4h per day for 3days; no mutations were introduced under normal growth conditions at 28°C. Surprisingly, however, mutations can be introduced without heat-shock treatment in rice. This might be due to the culture conditions, with relatively higher temperature (30°C or higher) and constant light during callus proliferation. Taken together, we demonstrated that SpCas12f can be used to achieve targeted mutagenesis in rice. SpCas12f is thus a useful tool for genome editing in rice and is suitable for virus vector-mediated genome editing due to its very small size.

Genome editing by introduction of Cas9/sgRNA into plant cells using temperature-controlled atmospheric pressure plasma.

Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT. The L-(I-SceI)-UC system allowed the detection of successful genome editing by measuring the chemiluminescent signal observed upon re-functionalization of the luciferase (LUC) gene following genome editing. Similarly, the sGFP-waxy-HPT system conferred hygromycin resistance caused by hygromycin phosphotransferase (HPT) during genome editing. CRISPR/Cas9 ribonucleoproteins targeting these reporter genes were directly introduced into rice calli or tobacco leaf pieces after treatment with N2 and/or CO2 plasma. Cultivation of the treated rice calli on a suitable medium plate produced the luminescence signal, which was not observed in the negative control. Four types of genome-edited sequences were obtained upon sequencing the reporter genes of genome-edited candidate calli. sGFP-waxy-HPT-carrying tobacco cells exhibited hygromycin resistance during genome editing. After repeated cultivation of the treated tobacco leaf pieces on a regeneration medium plate, the calli were observed with leaf pieces. A green callus that was hygromycin-resistant was harvested, and a genome-edited sequence in the tobacco reporter gene was confirmed. As direct introduction of the Cas9/sgRNA (single guide RNA) complex using plasma enables genome editing in plants without any DNA introduction, this method is expected to be optimized for many plant species and may be widely applied for plant breeding in the future.

Transcription factors NAC20 and NAC26 interact with RPBF to activate albumin accumulations in rice endosperm.

Transcription factors NAC20 and NAC26 interact with RPBF to activate albumin accumulations in rice endosperm.

Specific suppression of long terminal repeat retrotransposon mobilization in plants.

The tissue culture passage necessary for the generation of transgenic plants induces genome instability. This instability predominantly involves the uncontrolled mobilization of LTR retrotransposons (LTR-TEs), which are the most abundant class of mobile genetic elements in plant genomes. Here, we demonstrate that in conditions inductive for high LTR-TE mobilization, like abiotic stress in Arabidopsis (Arabidopsis thaliana) and callus culture in rice (Oryza sativa), application of the reverse transcriptase (RT) inhibitor known as Tenofovir substantially affects LTR-TE RT activity without interfering with plant development. We observed that Tenofovir reduces extrachromosomal DNA accumulation and prevents new genomic integrations of the active LTR-TE ONSEN in heat-stressed Arabidopsis seedlings, and transposons of O. sativa 17 and 19 (Tos17 and Tos19) in rice calli. In addition, Tenofovir allows the recovery of plants free from new LTR-TE insertions. We propose the use of Tenofovir as a tool for studies of LTR-TE transposition and for limiting genetic instabilities of plants derived from tissue culture.

Biochemical Characterization of Orange-Colored Rice Calli Induced by Target Mutagenesis of OsOr Gene.

We generated an orange-colored (OC) rice callus line by targeted mutagenesis of the orange gene (OsOr) using the CRISPR-Cas9 system. The OC line accumulated more lutein, β-carotene, and two β-carotene isomers compared to the WT callus line. We also analyzed the expression levels of carotenoid biosynthesis genes by qRT-PCR. Among the genes encoding carotenoid metabolic pathway enzymes, the number of transcripts of the PSY2, PSY3, PDS, ZDS and β-LCY genes were higher in the OC line than in the WT line. In contrast, transcription of the ε-LCY gene was downregulated in the OC line compared to the WT line. In addition, we detected increases in the transcript levels of two genes involved in carotenoid oxidation in the OC lines. The developed OC lines also showed increased tolerance to salt stress. Collectively, these findings indicate that targeted mutagenesis of the OsOr gene via CRISPR/Cas9-mediated genome editing results in β-carotene accumulation in rice calli. Accordingly, we believe that this type of genome-editing technology could represent an effective alternative approach for enhancing the β-carotene content of plants.

QTLs Related to Rice Callus Regeneration Ability: Localization and Effect Verification of qPRR3

Mature and efficient tissue culture systems are already available for most japonica rice varieties (Oryza sativa ssp. geng). However, it remains challenging to regenerate the majority of indica rice varieties (Oryza sativa ssp. xian). In this study, quantitative trait loci (QTLs) associated with rice callus regeneration ability were identified based on the plant regeneration rate (PRR) and total green plant rate (TGPR) of the 93-11 × Nip recombinant inbred line population. Significant positive correlations were found between PRR and TGPR. A total of three QTLs (one for PRR and two for TGPR) were identified. qPRR3 (located on chromosome 3) was detected for both traits, which could explain 13.40% and 17.07% of the phenotypic variations of PRR and TGPR, respectively. Subsequently, the effect of qPRR3 on callus regeneration ability was validated by cryptographically tagged near-isogenic lines (NILs), and the QTL was narrowed to an interval of approximately 160 kb. The anatomical structure observation of the regenerated callus of the NILs revealed that qPRR3 can improve the callus regeneration ability by promoting the regeneration of shoots.

Ectopic expression of WOX5 promotes cytokinin signaling and de novo shoot regeneration.

WOX5 has a potential in activating cytokinin signaling and shoot regeneration, in addition to its role in pluripotency acquisition. Thus, overexpression of WOX5 maximizes plant regeneration capacity during tissue culture. In vitro plant regeneration involves two steps: callus formation and de novo shoot organogenesis. The WUSCHEL-RELATED HOMEOBOX 5 (WOX5) homeodomain transcription factor is known to be mainly expressed during incubation on callus-inducing medium (CIM) and involved in pluripotency acquisition in callus, but whether WOX5 also affects de novo shoot regeneration on cytokinin-rich shoot-inducing medium (SIM) remains unknown. Based on the recent finding that WOX5 promotes cytokinin signaling, we hypothesized that ectopic expression of WOX5 beyond CIM would further enhance overall plant regeneration capacity, because intense cytokinin signaling is particularly required for shoot regeneration on SIM. Here, we found that overexpression of the WOX5 gene on SIM drastically promoted de novo shoot regeneration from callus with the repression of type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, negative regulators of cytokinin signaling. The enhanced shoot regeneration phenotypes were indeed dependent on cytokinin signaling, which were partially suppressed in the progeny derived from crossing WOX5-overexpressing plants with cytokinin-insensitive 35S:ARR7 plants. The function of WOX5 in enhancing cytokinin-dependent shoot regeneration is evolutionarily conserved, as conditional overexpression of OsWOX5 on SIM profoundly enhanced shoot regeneration in rice callus. Overall, our results provide the technical advance that maximizes in vitro plant regeneration by constitutively expressing WOX5, which unequivocally promotes both callus pluripotency and de novo shoot regeneration.

Issue Information

Plant Biotechnology JournalVolume 20, Issue 10 p. 1845-1846 Issue InformationOpen Access Issue Information First published: 21 September 2022 https://doi.org/10.1111/pbi.13625AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Front cover image: The cover image is the engineering of red betanin biosynthesis in N. benthamiana leaves (upper panel), transgenic rice calli (middle panel) and seedings (lower panel) using a novel multigene stacking system TGSII-UNiE. In this issue, Zhao et al develops a simple and fl exible TGSII-UNiE system for high-effi ciency assembly of long DNAs and multiple genes, which uses unique nucleotide sequence-guided nicking endonuclease (UNiE)-mediated DNA assembly (UNiEDA) method combined with improved Cre/loxP recombination-mediated TransGene Stacking II (TGSII) system. Due to its effi ciency, convenience and low price, the TGSII-UNiE system shows signifi cant application potential for plant functional genomics, synthetic biology, metabolic engineering. Cover illustration refers to the article in this issue (Zhao et al., 1983–1995). Volume20, Issue10October 2022Pages 1845-1846 RelatedInformation

A rice plant expressing viral glycoprotein NSvc2‐NS reduces the transmission of rice stripe virus by the small brown planthopper

Plant viruses transmitted by arthropod vectors threaten crop health worldwide. Rice stripe virus (RSV) is one of the most important rice viruses in East Asia and is transmitted by the small brown planthopper (SBPH). Previously, it was demonstrated that the viral glycoprotein NSvs2-N could mediate RSV infection of the vector midgut. Therefore, NSvc2-N protein could potentially be used to reduce RSV transmission by competitively blocking midgut receptors. Here, we report that transgenic rice plants expressing viral glycoprotein can interfere with RSV acquisition and transmission by SBPH. The soluble fraction (30-268 amino acids, designated NSvs2-NS ) of NSvs2-N was transformed into rice calli, which produced plants harboring the exogenous gene. When SBPH was fed on transgenic plants prior to RSV-infected rice (sequential feeding) and when insects were fed on RSV-infected transgenic plants (concomitant feeding), virus acquisition by the insect vector was inhibited, and subsequent viral titers were reduced. Immunofluorescence labeling also indicated that viral infection of the insect midgut was inhibited after SBPH was fed on transgenic plants. The system by which RSV infected insect cells in vitro was used to corroborate the role of NSvc2-NS in reducing viral infection. After the cells were incubated with transgenic rice sap, the virus infection rate of the cells decreased significantly, and viral accumulation in the cells was lower than that in the control group. These results demonstrated the negative effect of NSvs2-NS transgenic plants on RSV transmission by insect vectors, which provides a novel and effective way to control plant viral diseases. © 2022 Society of Chemical Industry.

Overexpression of maize GOLDEN2 in rice and maize calli improves regeneration by activating chloroplast development.

Golden2 (G2), a member of the GARP transcription factor superfamily, regulates several biological processes and phytohormone signaling pathways in plants. In this study, we used a rice codon-optimized maize G2 gene (rZmG2) to improve the regeneration efficiency of rice and maize calli for genetic transformation. We isolated a promoter driving strong and callus-specific expression from rice to drive rZmG2 transcription from a transgene after transformation of two indica and two japonica rice cultivars. The resulting rZmG2 transgenic calli turned green in advance at the differentiation stage, thus significantly raising the regeneration rates of the transgenic indica and japonica rice plants relative to control transformations. Similar effect of this gene on improving maize transformation was also observed. Transcriptome sequencing and RT-qPCR analyses showed that many rice genes related to chloroplast development and phytohormones are upregulated in rZmG2-transgenic calli. These results demonstrate that rZmG2 can promote embryogenic callus differentiation and improve regeneration efficiency by activating chloroplast development and phytohormone pathways. We also established a heat-inducible Cre/loxP-based gene-excision system to remove rZmG2 and the antibiotic selectable gene after obtaining the transgenic plants. This study provides a useful tool for functional genomics work and biotechnology in plants.

Host induced gene silencing of Magnaporthe oryzae by targeting pathogenicity and development genes to control rice blast disease.

Rice blast disease caused by the hemi-biotrophic fungus Magnaporthe oryzae is the most destructive disease of rice world-wide. Traditional disease resistance strategies for the control of rice blast disease have not proved durable. HIGS (host induced gene silencing) is being developed as an alternative strategy. Six genes (CRZ1, PMC1, MAGB, LHS1, CYP51A, CYP51B) that play important roles in pathogenicity and development of M. oryzae were chosen for HIGS. HIGS vectors were transformed into rice calli through Agrobacterium-mediated transformation and T0, T1 and T2 generations of transgenic rice plants were generated. Except for PMC1 and LHS1, HIGS transgenic rice plants challenged with M. oryzae showed significantly reduced disease compared with non-silenced control plants. Following infection with M. oryzae of HIGS transgenic plants, expression levels of target genes were reduced as demonstrated by Quantitative RT-PCR. In addition, treating M. oryzae with small RNA derived from the target genes inhibited fungal growth. These findings suggest RNA silencing signals can be transferred from host to an invasive fungus and that HIGS has potential to generate resistant rice against M. oryzae.