Advancing Crop Transformation in the Era of Genome Editing.

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized.

Molecular and genetic aspects of controlling the soilborne necrotrophic pathogens Rhizoctonia and Pythium.

The soilborne necrotrophic pathogens Rhizoctonia and Pythium infect a wide range of crops in the US and worldwide. These pathogens pose challenges to growers because the diseases they cause are not adequately controlled by fungicides, rotation or, for many hosts, natural genetic resistance. Although a combination of management practices are likely to be required for control of Rhizoctonia and Pythium, genetic resistance remains a key missing component. This review discusses the recent deployment of introduced genes and genome-based information for control of Rhizoctonia, with emphasis on three pathosystems: Rhizoctonia solani AG8 and wheat, R. solani AG1-IA and rice, and R. solani AG3 or AG4 and potato. Molecular mechanisms underlying disease suppression will be addressed, if appropriate. Although less is known about genes and factors suppressive to Pythium, pathogen genomics and biological control studies are providing useful leads to effectors and antifungal factors. Prospects for resistance to Rhizoctonia and Pythium spp. will continue to improve with growing knowledge of pathogenicity strategies, host defense gene action relative to the pathogen infection process, and the role of environmental factors on pathogen-host interactions.

The wheat HMW-glutenin 1Dy10 gene promoter controls endosperm expression in Brachypodium distachyon.

The grass species Brachypodium distachyon has emerged as a model system for the study of gene structure and function in temperate cereals. As a first demonstration of the utility of Brachypodium to study wheat gene promoter function, we transformed it with a T-DNA that included the uidA reporter gene under control of a wheat High-Molecular-Weight Glutenin Subunit (HMW-GS) gene promoter and transcription terminator. For comparison, the same expression cassette was introduced into wheat by biolistics. Histochemical staining for ß-glucuronidase (GUS) activity showed that the wheat promoter was highly expressed in the endosperms of all the seeds of Brachypodium and wheat homozygous plants. It was not active in any other tissue of transgenic wheat, but showed variable and sporadic activity in a minority of styles of the pistils of four homozygous transgenic Brachypodium lines. The ease of obtaining transgenic Brachypodium plants and the overall faithfulness of expression of the wheat HMW-GS promoter in those plants make it likely that this model system can be used for studies of other promoters from cereal crop species that are difficult to transform.

The LP2 leucine-rich repeat receptor kinase gene promoter directs organ-specific, light-responsive expression in transgenic rice.

Biotechnologists seeking to limit gene expression to nonseed tissues of genetically engineered cereal crops have only a few choices of well characterized organ-specific promoters. We have isolated and characterized the promoter of the rice Leaf Panicle 2 gene (LP2, Os02g40240). The LP2 gene encodes a leucine-rich repeat-receptor kinase-like protein that is strongly expressed in leaves and other photosynthetic tissues. Transgenic rice plants containing an LP2 promoter-GUS::GFP bifunctional reporter gene displayed an organ-specific pattern of expression. This expression corresponded to transcript levels observed on RNA blots of various rice organs and microarray gene expression data. The strongest beta-glucuronidase activity was observed in histochemically stained mesophyll cells, but other green tissues and leaf cell types including epidermal cells also exhibited expression. Low or undetectable levels of LP2 transcript and LP2-mediated reporter gene expression were observed in roots, mature seeds, and reproductive tissues. The LP2 promoter is highly responsive to light and only weak expression was detected in etiolated rice seedlings. The specificity and strength of the LP2 promoter suggests that this promoter will be a useful control element for green tissue-specific expression in rice and potentially other plants. Organ-specific promoters like LP2 will enable precise, localized expression of transgenes in biotechnology-derived crops and limit the potential of unintended impacts on plant physiology and the environment.

Effects of overexpression of high molecular weight glutenin subunit 1Dy10 on wheat tortilla properties.

Wheat (Triticum aestivum L.) flour properties necessary for optimal tortilla production have not been identified. Transgenic wheats (Triticum aestivum L.) overexpressing high molecular weight glutenin subunit (HMW-GS) 1Dy10 were used to make tortilla and their quality was evaluated. The level of HMW-GS 1Dy10 in flours derived from transgenic wheats was 2.5-5.8-fold greater than in controls. Polymeric proteins in the transgenic samples had a molecular weight distribution shifted toward larger polymers as indicated by increased levels of polymeric proteins present and greater M(w) averages of the largest fractions in the insoluble polymeric proteins. Dough derived from transgenic wheats had greater resistance to extension and lower extensibility than controls. Tortilla quality evaluation revealed that tortillas originated from transgenic wheats had decreased diameter, greater thickness and rupture force, and lower rollability scores and stretchability than controls. The presence of 1RS chromosomal translocations from rye (Secale cereale L.) in transgenic wheat decreased the negative effects of overexpression of HMW-GS 1Dy10, as tortillas made with this flour mostly exhibited quality properties similar to those made from control flour. Results suggested that the negative effects of overexpression of HMW-GS 1Dy10 on tortilla properties were derived from a nonideal gluten matrix formation.

The expression of a bean PGIP in transgenic wheat confers increased resistance to the fungal pathogen Bipolaris sorokiniana.

A possible strategy to control plant pathogens is the improvement of natural plant defense mechanisms against the tools that pathogens commonly use to penetrate and colonize the host tissue. One of these mechanisms is represented by the host plant's ability to inhibit the pathogen's capacity to degrade plant cell wall polysaccharides. Polygalacturonase-inhibiting proteins (PGIP) are plant defense cell wall glycoproteins that inhibit the activity of fungal endopolygalacturonases (endo-PGs). To assess the effectiveness of these proteins in protecting wheat from fungal pathogens, we produced a number of transgenic wheat lines expressing a bean PGIP (PvPGIP2) having a wide spectrum of specificities against fungal PGs. Three independent transgenic lines were characterized in detail, including determination of the levels of PvPGIP2 accumulation and its subcellular localization and inhibitory activity. Results show that the transgene-encoded protein is correctly secreted into the apoplast, maintains its characteristic recognition specificities, and endows the transgenic wheat with new PG recognition capabilities. As a consequence, transgenic wheat tissue showed increased resistance to digestion by the PG of Fusarium moniliforme. These new properties also were confirmed at the plant level during interactions with the fungal pathogen Bipolaris sorokiniana. All three lines showed significant reductions in symptom progression (46 to 50%) through the leaves following infection with this pathogen. Our results illustrate the feasibility of improving wheat's defenses against pathogens by expression of proteins with new capabilities to counteract those produced by the pathogens.