Invented by Vipula Kiran Shukla, Holly Jean Butler, Anthony Thinh Ngoc Trieu, Aaron Todd Woosley, Pamela Rene Haygood, Christie Min Dewes, James Patrick Connell, Ignacio Mario Larrinua, Zihua Hu, Avutu Sambi Reddy, Corteva Agriscience LLC

The market for nucleic acid compositions conferring disease resistance is experiencing significant growth due to the increasing demand for sustainable and environmentally friendly agricultural practices. Nucleic acid compositions, such as RNA interference (RNAi) and gene editing technologies, have emerged as powerful tools in developing disease-resistant crops. Diseases caused by pathogens, pests, and viruses pose a significant threat to global food security. Traditional methods of disease control, such as chemical pesticides and fungicides, have proven to be harmful to the environment and human health. As a result, there is a growing need for alternative solutions that can effectively combat diseases while minimizing the negative impacts on the ecosystem. Nucleic acid compositions offer a promising approach to disease resistance in crops. RNAi, for instance, involves the introduction of small RNA molecules that can specifically target and silence the genes responsible for disease susceptibility. By suppressing the expression of these genes, RNAi can effectively enhance the plant’s immune response and reduce its vulnerability to diseases. Gene editing technologies, such as CRISPR-Cas9, have also revolutionized the field of disease resistance. These tools enable scientists to precisely modify the plant’s genetic material, introducing specific changes that confer resistance to diseases. By editing genes responsible for disease susceptibility, researchers can develop crops that are naturally resistant to pathogens and pests. The market for nucleic acid compositions conferring disease resistance is driven by several factors. Firstly, the increasing global population and changing dietary preferences have led to a higher demand for food production. Disease outbreaks can significantly reduce crop yields, leading to food shortages and price fluctuations. Disease-resistant crops can help ensure a stable and secure food supply, making them an attractive option for farmers and food producers. Secondly, the growing awareness of the environmental and health impacts of chemical pesticides has prompted a shift towards sustainable agricultural practices. Nucleic acid compositions offer a targeted and precise approach to disease control, reducing the need for harmful chemicals. This aligns with the increasing consumer demand for organic and environmentally friendly products. Furthermore, advancements in biotechnology and genetic engineering have made nucleic acid compositions more accessible and cost-effective. The development of efficient delivery systems and improved understanding of plant genetics have accelerated the commercialization of disease-resistant crops. This has opened up opportunities for both large agribusinesses and smaller biotech companies to enter the market and develop innovative solutions. However, challenges remain in the widespread adoption of nucleic acid compositions for disease resistance. Regulatory frameworks and public acceptance of genetically modified organisms (GMOs) vary across countries, which can hinder the commercialization of genetically modified crops. Additionally, concerns about potential unintended consequences and long-term effects on ecosystems need to be addressed through rigorous testing and risk assessment. In conclusion, the market for nucleic acid compositions conferring disease resistance is poised for significant growth as the demand for sustainable and environmentally friendly agricultural practices continues to rise. RNAi and gene editing technologies offer powerful tools to develop disease-resistant crops, ensuring a stable and secure food supply while minimizing the negative impacts on the environment. However, regulatory challenges and public acceptance remain important considerations in the widespread adoption of these technologies.

The Corteva Agriscience LLC invention works as follows

This invention involves the identification and isolation genes conferring disease control properties to plants as well as the plants containing such genes. These genes were derived from Nicotiana benthamiana and Oryzae Sativa (var. Indica IR7, Papaver Rhoeas (Rifai 1295-22), Saccharomyces Cervisiae, and Trichoderma Harzianum are the genes that were used to create this product. The control is conferred against one or more phytopathogens, including: Aspergillus flvus, Cercospora zeaemaydis (Rifai 1295-22), Fusarium monilforme (Rifai 1295-22), Fusarium graminearum (Rifai 1295-22), Helminthosporium maydis (Rifai 1205-22), Phoma lingam and Phomopsis helianthi This invention also encompasses homologous and heterologous peptide sequences that have a high degree similarity in function.

Background for Nucleic Acid Compositions conferring Disease Resistance

Since the dawn of agriculture, thousands of years ago farmers have been fighting to reduce the impact of pests on crops. The spread of plant diseases by bacteria and fungi is a major cause for crop production to be limited worldwide. In the US, for example, head blight caused by the fungi Fusarium poae and Fusarium Graminearum caused an estimated $3 billion loss in wheat and barley between 1991-1996. In less developed countries where food production is typically at or below the level of sustenance, the impact is far more severe. The impact of diseases is not limited to the overall yield. They also have an effect on the quality. The destruction of crops, and the resulting decrease in the quality of food produced has a profound impact on the socioeconomic situation. This is illustrated by the Irish Potato Famine of 1800 and the subsequent mass starvation. Some plant pathogens can also be harmful to human health, like mycotoxins, which are produced by fungi phytopathogens.

Fungi is a diverse and versatile group that can successfully inhabit most natural habitats. Fewer than 10% of all the known fungi species are capable of colonizing plants. Of these, only a fraction is responsible for plant disease. Virtually all flowering plants can be attacked and are susceptible to some phytopathogenic fungus. The specificity of the interactions is determined both by host range restrictions of plant and microbe. Fungal phytopathogens can be divided into three main categories: 1) opportunistic pathogens with a wide host range, but low virulence; 2) facultative pathogens which require living plants for growth, but are able to survive without them in certain circumstances; and 3) obligate parasites for whom a living plant is a requirement for survival. The second group of phytopathogens, the facultative parasites, is the one that most interests agricultural researchers. This is because many of the most dangerous and virulent disease agents are found in this class. Fungal phytopathogens are responsible for many important agronomically relevant diseases, including: leaf blotch and glume, stalk/head decay, leaf spot and blight on rice, and corn smut.

Plant Defense

Over the course of natural selection and evolution, plants have developed a number of mechanisms to defend themselves against phytopathogens. This process is often called the “evolutionary arms race”. The rapidity with which most pathogens can overcome plant defenses is what keeps the process going. Plants can mount generalized or specific responses to disease organisms using a variety of defensive mechanisms. Plants can protect themselves from disease organisms by using defense mechanisms like bark, trichomes, and waxy cuticles. Some plants also secrete compounds such as gums and resins that act not only as a barrier against pathogens but can sometimes be repellent.

Plants have the ability to mount defenses when faced with pathogens. In order to mount these induced responses, the plant must recognize the pathogen, activate a defense mechanism, and localize infection in order to prevent invasion/spread and full-blown diseases. Incompatible interactions are described when the pathogen cannot successfully infect and parasitize the host plant. The study of incompatible interactions has been made easier by the molecular analysis of plant and microbe gene identified through various mutant screens. In general, defense pathways that are induced by incompatible interactions can be divided into two groups: hypersensitive response (HR) and systemic acquired resistant (SAR). It is evident that these pathways have many branch points which intersect and overlap.

The HR is characterized by localized cell death induced in the host plant near the pathogen invasion site. The HR is often associated with the formation of necrotic flecks that contain dead plant cells, within hours of contact with pathogens. This cell death prevents the pathogen from gaining access to nutrients, which causes the pathogen to be arrested and protects the rest of plant against the disease agent. The activation of programmed death (apoptosis), by the plant, and/or the switch in cell metabolism of the plant are the mechanisms of HR. These biochemical pathways produce compounds that are toxic to pathogens and plants. The presence of reactive species, such as superoxide anion and H2O2, can trigger HR. They also act as signal molecules and are converted into highly reactive and destructive oxygen radicals. The induction of HR is also linked to the benzoic acid, salicylic acid, and their respective glucosides conjugates. These molecules also have signalling roles and can be directly antimicrobial as well as a number of classes of PR proteins.

SAR is an inducible, broad-spectrum plant immunity that is induced after the formation a necrotic lesions, either as part of HR, or as a disease symptom. It is not restricted to incompatible interactions but can be induced through compatible interactions with microbes that cause disease. This resistance or immunity spreads throughout the plant and is developed in distant, unchallenged areas. SAR is nonspecific and acts throughout the plant. It reduces disease symptoms, even those caused by highly virulent pathogens. The elicitor SA can induce it in a dose-dependent way. It involves a complex group of downstream elicitors and signal transduction molecules. The SAR response can be characterized by the coordinated induction of genes from several families in uninfected leaf tissue, such as chitinases and?-1.3% glucanases. PR-1 proteins are also involved. “The exact mechanisms of SAR, HR and plant disease resistance are still being studied.

Traditional Agricultural Methods for Plant Disease Control

Over the course of several centuries, farmers have developed methods to control plant diseases. Romans used farming techniques like crop rotation, controlled irrigation and manure application. These methods alone are not effective enough to combat diseases by modern standards. They are still standard practices and they contribute to any comprehensive pest control program.

Breeding methods, in addition to husbandry have been used to develop disease resistant cultivars. Plant breeders can now take advantage of genetic variations and induced mutations by selecting and propagating cultivars that contain desirable traits. Breeders have a variety of genetic techniques and methods at their disposal, such as embryo rescue, cell-fusion, and mutagenesis. Breeders’ programs are based on the cultivars they wish to improve, e.g. hybrids vs. purebreds, and the reproductive biology (self-pollinated or out-crossed) of each species. A successful breeding program was the introduction of traits from a Mexican specie into more than 50% of all cultivars. This resulted in blight resistant potatoes. The conventional breeding methods will continue to be important in improving agricultural crops. However, they may not meet the increasing demand for many agronomic characteristics, including disease resistance. The ability of pathogens, to quickly overcome resistance bred in new races of plants, limits the useful life of these crops.

In the past few decades, agriculture techniques have evolved to include a widespread and intensive use chemicals. In a recent study, the US agricultural sector spent $8.83 billion on pesticides in 1997. This was for 11 major crops. This is a large portion of the US agricultural economy. Bio-control has gained some farmers’ attention, though it is still a relatively small part of the industry. It is essential that new agricultural methods are developed to address the growing concern about global health and the environment.

AgBiotech Approaches for Plant Disease Control

The advent of modern biology has created new opportunities for research and practice in disease control. Scientists are focusing on the use of recombinant (rDNA), which allows new plant varieties to be created much faster than conventional breeding. rDNA methods allow for the introduction of genes into crop plants from distantly-related species, or even different biological kingdoms. This confers traits that offer significant agronomic benefits. Further, a detailed understanding of the traits introduced, including cellular function and location, can result in less variability and fine-tuning secondary effects. “Conventional breeding techniques can be used after a transgenic trait is introduced to a plant. This allows the hybridization of this transgenic line with useful germplasms and varieties, leading to crops that have many advantageous properties.

Agricultural Biotechnology (AgBiotech), approaches to disease resistant are usually three-fold. In order to bio-engineer the disease, researchers first analyze specific crops that have compatible interactions (causing diseases) with pathogens. Researchers then look for exogenous (other species/sources) factors that can be produced by crop plants to protect them from phytopathogens. Finaly, researchers are working to hyper-activate a plant’s defense response in order to give crops broad-spectrum protection against multiple disease agents at once. These approaches each have their own advantages and disadvantages. They’ve had limited success so far. Between 1987 and May 2000, there were 272 privately-sponsored trials that tested genes for resistance to fungal diseases in transgenic plants. 61 of these field trials were publicly-sponsored. Monsanto Company described a recent example of successful bioengineered disease-resistance product. It demonstrated that a potato engineered with an alfalfa peptide antifungal (defensin), showed robust resistance against the fungal pathogen Verticillium dahliae.

As AgBiotech accelerates into the genomics era and the post-genomics age, massive amounts of data are being collected to guide the search for genes which can be used with recombinant techniques. Transgenic technology is also overcoming its speed-limiting hurdles, allowing the expression and modulation multiple genes at once in transgenic plants. The informational and technical tools that agricultural biotechnologists have access to are improving and will continue to improve the speed of product development and discovery in relation to disease resistance. Many AgBiotech products are expected to enter the market as the regulatory and commercial frameworks are developed. It is reasonable to expect, therefore, that bioengineered plants will soon be part of an integrated, comprehensive disease management program across the entire agricultural enterprise.

Accordingly, what’s needed in the field are gene and polypeptides sequences that cause resistance to plant pathogens in plants, seeds, tissues, and/or cells.

This invention relates deoxyribonucleic (DNA) and amino acids sequences that confer disease-resistance phenotypes to plants as well as disease-resistant plants, plant seeds and tissues, and plant cells containing such sequences.

In some embodiments of the invention, polynucleotides or polypeptides are provided that confer disease resistance traits when expressed in plants. The present invention does not limit itself to any specific polynucleotide or polypeptide sequences that confer disease resistant phenotypes. In fact, many sequences can be used. In some embodiments, the present invention provides a nucleic sequence that can hybridize with any of SEQ NOs: 1 to 2318, and the nucleic acids that are compatible under low stringency. The expression of this isolated nucleic in a plant will result in a disease resistant phenotype. The present invention also provides vectors containing the polynucleotide sequencing. In yet other embodiments, these sequences can be operably linked with an exogenous promotor, most commonly a plant-promoter. The present invention does not limit itself to a particular promoter. In fact, a wide range of promoters are contemplated. These include, but are not limited to: 35S and19S of Cauliflower Vein Mosaic Virus; ubiquitin; heat shock and Rubisco promoters. In some embodiments the nucleic acids of the invention are arranged sense-orientation, while in others, they are arranged antisense-orientation in the vector. In yet other embodiments, the invention also provides a plant that contains one of the nucleic acids sequences or vectors mentioned above, along with seeds, leaves and fruit. In certain embodiments that are particularly preferred, the invention provides one or more of the nucleic acid sequences to be used in conferring disease resistance in plants.

In still other embodiments of the invention, processes are provided for making transgenic plants, which include providing a vector described above, a plant and transfecting it with the vector. The present invention also provides processes to confer a pathogen or disease resistance phenotype on a population of plants or a particular plant. This involves providing the vector described above, the plant and then transfecting it with the vector in conditions that allow the expression of the isolated RNA from the vector. In yet further embodiments, the invention provides an isolate nucleic acids selected from a group consisting SEQ ID Nos:1-2318 as well as nucleic sequences that can hybridize with any of them under conditions of low strictness for use in producing disease or pathogen-resistant plants. Other embodiments of the invention provide an isolated nucleic acids, compositions or vectors substantially as described in the claims or examples.

Brief Description of the Tables

The contig sequences are listed in Table 1. They correspond to the SEQ ID Nos:1-407, and 2256-2318.

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