CRISPR/Cas9-Based Gene Editing Using Egg Cell-Specific Promoters in Arabidopsis and Soybean.

CRISPR/Cas9-based systems are efficient genome editing tools in a variety of plant species including soybean. Most of the gene edits in soybean plants are somatic and non-transmissible when Cas9 is expressed under control of constitutive promoters. Tremendous effort, therefore, must be spent to identify the inheritable edits occurring at lower frequencies in plants of successive generations. Here, we report the development and validation of genome editing systems in soybean and Arabidopsis based on Cas9 driven under four different egg-cell specific promoters. A soybean ubiquitin gene promoter driving expression of green fluorescent protein (GFP) is incorporated in the CRISPR/Cas9 constructs for visually selecting transgenic plants and transgene-evicted edited lines. In Arabidopsis, the four systems all produced a collection of mutations in the T2 generation at frequencies ranging from 8.3 to 42.9%, with egg cell-specific promoter AtEC1.2e1.1p being the highest. In soybean, function of the gRNAs and Cas9 expressed under control of the CaMV double 35S promoter (2x35S) in soybean hairy roots was tested prior to making stable transgenic plants. The 2x35S:Cas9 constructs yielded a high somatic mutation frequency in soybean hairy roots. In stable transgenic soybean T1 plants, AtEC1.2e1.1p:Cas9 yielded a mutation rate of 26.8%, while Cas9 expression driven by the other three egg cell-specific promoters did not produce any detected mutations. Furthermore, the mutations were inheritable in the T2 generation. Our study provides CRISPR gene-editing platforms to generate inheritable mutants of Arabidopsis and soybean without the complication of somatic mutagenesis, which can be used to characterize genes of interest in Arabidopsis and soybean.

Virus-Induced Gene Silencing and Transient Gene Expression in Soybean (Glycine max) Using Bean Pod Mottle Virus Infectious Clones.

Virus-induced gene silencing (VIGS) is a powerful and rapid approach for determining the functions of plant genes. The basis of VIGS is that a viral genome is engineered so that it can carry fragments of plant genes, typically in the 200 to 300 base pair size range. The recombinant viruses are used to infect experimental plants, and wherever the virus invades, the target gene or genes will be silenced. VIGS is thus transient, and in the span of a few weeks, it is possible to design VIGS constructs and then generate loss-of-function phenotypes through RNA silencing of the target genes. In soybean (Glycine max), the Bean pod mottle virus (BPMV) has been engineered to be valuable tool for silencing genes with diverse functions and also for over-expression of foreign genes. This protocol describes a method for designing BPMV constructs and using them to silence or transiently express genes in soybean. © 2016 by John Wiley & Sons, Inc.

Differential requirement of ribosomal protein S6 by plant RNA viruses with different translation initiation strategies.

Potyvirus infection has been reported to cause an increase in the mRNA transcripts of many plant ribosomal proteins (r-proteins). In this study, increased expression of r-protein mRNA transcripts was determined to occur in Nicotiana benthamiana during infection by potyviruses as well as a tobamovirus demonstrating that this response is not unique to potyviruses. Five r-protein genes, RPS6, RPL19, RPL13, RPL7, and RPS2, were silenced in N. benthamiana to test their roles in viral infection. The accumulation of both Turnip mosaic virus (TuMV), a potyvirus, and Tobacco mosaic virus (TMV), a tobamovirus, was dependent on RPL19, RPL13, RPL7, and RPS2. However, TMV was able to accumulate in RPS6-silenced plants while accumulation of TuMV and Tomato bushy stunt virus (TBSV) was abolished. These results demonstrate that cap-independent TuMV and TBSV require RPS6 for their accumulation, whereas accumulation of TMV is independent of RPS6.

Distinct biphasic mRNA changes in response to Asian soybean rust infection.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is now established in all major soybean-producing countries. Currently, there is little information about the molecular basis of ASR-soybean interactions, which will be needed to assist future efforts to develop effective resistance. Toward this end, abundance changes of soybean mRNAs were measured over a 7-day ASR infection time course in mock-inoculated and infected leaves of a soybean accession (PI230970) carrying the Rpp2 resistance gene and a susceptible genotype (Embrapa-48). The expression profiles of differentially expressed genes (ASR-infected compared with the mock-inoculated control) revealed a biphasic response to ASR in each genotype. Within the first 12 h after inoculation (hai), which corresponds to fungal germination and penetration of the epidermal cells, differential gene expression changes were evident in both genotypes. mRNA expression of these genes mostly returned to levels found in mock-inoculated plants by 24 hai. In the susceptible genotype, gene expression remained unaffected by rust infection until 96 hai, a time period when rapid fungal growth began. In contrast, gene expression in the resistant genotype diverged from the mock-inoculated control earlier, at 72 h, demonstrating that Rpp2-mediated defenses were initiated prior to this time. These data suggest that ASR initially induces a nonspecific response that is transient or is suppressed when early steps in colonization are completed in both soybean genotypes. The race-specific resistance phenotype of Rpp2 is manifested in massive gene expression changes after the initial response prior to the onset of rapid fungal growth that occurs in the susceptible genotype.