Plant virus resistance genes

For simplicity, only one generic Cas nuclease is drawn. The viral genome forms double-stranded intermediates during replication inside the host cell nuclei. One potential caveat of this approach is that alterations to the viral DNA sequence may occur near the cleavage target due to DNA repair within the host cell.

Among all the guide RNAs used against TYLCV, the ones targeting the stem-loop sequence within the replication of origin were the most effective, possibly because of the reduced occurrence of viable escapee variants of the virus with mutations in this region Ali et al. Using Cas13a to target viral RNA substrates does not induce DNA breakage and thus would not introduce undesired off-target mutations to the host genome Abudayyeh et al.

Although a field test of CRISPR-Cas-based antiviral resistance on crop species has not been reported, the laboratory studies have demonstrated its potential as an antiviral tool. To ensure durable resistance, it is important to consider potential viral evasions from the surveillance by the specific guide RNA used. Choosing genomic targets essential for the replication or movement of the viral pathogen minimizes viral evasions Ali et al.

Multiplexing the guide RNAs can also improve the robustness. In addition, it has been hypothesized that the use of Cas12a also known as Cpf1 may reduce the occurrence of escapee viral variants because mutations caused by CRISPR-Cas12a are less likely to abloish the recognition of the target by the orginal guide RNA Ali et al.

When both the R gene in the plant host and the cognate avr in the pathogen are present, the plant-pathogen interaction is incompatible and the host exhibits full resistance to the pathogen Flor, The effectiveness of R gene-mediated resistance was first demonstrated by British scientist Rowland Biffen in wheat Triticum sp.

Since that time, numerous R genes have been cloned and introduced into varieties of the same species De Wit et al. For example, the introduction of the maize Zea mays R gene Rxo1 into rice Oryza sativa conferred resistance to the bacterial streak pathogen Xanthomonas oryzae pv. In tomato, multiyear fields trials under commercial type growth conditions have demonstrated that tomatoes expressing the pepper Bs2 R gene confer robust resistance to Xanthomonas sp. Wheat transgenically expressing various alleles of the wheat resistance locus Pm3 exhibited race-specific resistance to powdery mildew in the field Brunner et al.

Potatoes transgenically expressing wild potato R gene RB or Rpi-vnt1. Notably, transgenic potato expressing Rpi-vnt1. Simplot is to date the only case of a genetically engineered crop with enhanced resistance to a nonviral pathogen that has been approved for commercial use Table 1. Successful pathogens often evade detection by host R genes Jones and Dangl, Thus, disease resistance conferred by a single R gene often lacks durability in the field because pathogens can evolve to evade recognition by mutating the corresponding avr gene.

For improved durability and to broaden the resistance spectrum, multiple R genes are often introduced simultaneously, which is commonly known as stacking Li et al. Resistance conferred by stacked R genes is predicted to be long lasting, as the evolution of a pathogen strain that could overcome resistance conferred by multiple R genes simultaneously is a low occurrence event.

One approach to stack R genes is by cross breeding preexisting R loci Fig. Breeders can then use marker-assisted selection to identify the progeny with the desired R gene composition Das and Rao, For example, bacterial blight in rice, caused by the bacterial pathogen Xanthomonas oryzae pv.

Through cross breeding and marker-assisted selection, three R genes that confer resistance to bacterial blight in rice, Xa21 , Xa5 , and Xa13 were introduced into a deep-water rice cultivar called Jalmagna Pradhan et al. The resulting line with the stacked R genes exhibited a higher level of field resistance to eight Xoo isolates tested Pradhan et al.

Although marker-assisted selection has largely improved the efficiency of the selection process, combining multiple loci through this approach can still be highly time consuming. Methods of R gene stacking. A, Stacking by marker-assisted breeding is performed by cross pollinating individuals with existing trait loci followed by marker-assisted selection for progeny with combined trait loci.

B, Stacking can be completed by combining multiple genes into a single stack vector and introducing them together as a single transgenic event. C, Stacking by targeted insertion aims at placing new gene s adjacent to an existing locus. This process can be performed iteratively to stack large numbers of genes.

In B and C, the stacked genes are genetically linked and thus can be easily introduced into a different genetic background as a single locus through breeding. As an alternative to gene stacking through marker-assisted selection, scientists can assemble multiple R gene cassettes onto one plasmid and then introduce this R gene cluster en bloc at a single genetic locus through plant transformation Fig.

This approach, called molecular stacking, simplifies the selection process, as all the R genes introduced this way are inherited as a single genetic locus. As an example of molecular stacking, Zhu et al.

The resulting triple gene transformants displayed broad-spectrum resistance equivalent to the sum of the strain-specific resistance conferred by all three individual Rpi genes under greenhouse conditions Zhu et al.

In a related study, a single DNA fragment harboring Rpi-vnt1. The R gene-stacked lines showed broad-spectrum late blight resistance because of the introduction of both R genes Jo et al. Resistance to the late blight pathogen in both the double gene-stacked and the triple gene-stacked potatoes mentioned above was confirmed under field conditions Haverkort et al. Similarly, in a field study of two African highland potato varieties in Uganda, Ghislain et al.

The yields of these R gene stacked potato varieties were three times higher than the national average. These results demonstrate that resistance achieved by this strategy does not negatively affect yield Ghislain et al. The above studies show the simplicity and effectiveness of molecular stacking for engineering broad-spectrum disease resistance, especially in vegetatively propagated crop species, where breeding stacks are not practical.

Despite the advantages of molecular stacking, the number of genes that can be introduced through molecular stacking is often restrained by the limit in the length of the DNA insert that can be put into a vector Que et al. This limitation can be overcome if DNA fragments can be sequentially inserted at the same genomic target Fig. Recent breakthroughs in genome-editing technologies in plants enable such targeted insertion of DNA fragments in diverse crop species Puchta and Fauser, ; Rinaldo and Ayliffe, ; Kumar et al.

This technology allows multiple R gene cassettes to be inserted at a single locus in multiple rounds Ainley et al. As genome-editing platforms in plants continue to be improved, the efficiency of targeted insertion, the size limit of the DNA inserts, and the number of applicable plant species are increasing. Future advancements in targeted gene insertion would offer new opportunities for stacking of large numbers of R genes and engineered viral resistance at a single locus for broad-spectrum, durable disease resistance and convenience in breeding.

Plants possess immune receptors that perceive pathogens and trigger cellular defense responses. Discovery of novel immune receptors recognizing major virulence factors will enrich the repertoire of known immune receptor genes that may be deployed in the field.

The nucleotide-binding leucine-rich repeats NLR family proteins comprise a major category of intracellular immune receptors conserved across the plant and animal kingdoms Maekawa et al.

A distinct hallmark for NLR-mediated defense is the onset of localized programmed cell death known as the hypersensitive response, which plays a crucial role in restricting the movement of pathogens del Pozo and Lam, ; Pontier et al. The hypersensitive response is often used as a marker to screen diverse plant germplasm for novel functional NLRs recognizing known effectors Vleeshouwers and Oliver, During the screening, core virulence factors are delivered into plant leaves either as transiently expressed genes through Agro-infiltration Du et al.

Once a collection of germplasm exhibiting various degrees of resistance to a particular pathogen strain is identified, comparative genomics tools such as resistance gene enrichment sequencing RenSeq; Jupe et al. This leads to the cloning of new NLR genes and their potential deployment in crop protection through genetic engineering. For example, RenSeq was successfully applied in the accelerated identification and cloning of an anti- P.

Transgenic expression of Rpi-amr3i in potato conferred full resistance to P. In a more recent study, Chen et al. In addition to its application in potato research, RenSeq has also been employed in the cloning of wheat NLR resistance genes against the stem rust fungus Puccinia graminis f. As an example, Arora et al.

This led to the rapid cloning of four stem rust Sr resistance genes Arora et al. In a related study, Steuernagel et al. The study revealed the identity of two stem rust NLR genes, Sr22 and Sr45 , which confer resistance to commercially important races of the stem rust pathogen Steuernagel et al. Although future field experiments are required to evaluate the potential of the deployment of these newly identified NLR genes, the above lab studies demonstrate that RenSeq is a powerful tool to rapidly identify novel NLR genes.

In addition to the identification of useful immune receptors from diverse plant germplasm, researchers have attempted to engineer known immune receptors for new ligand specificity.

For example, the fusion of the ectodomain of the Arabidopsis pattern recognition receptor EF-Tu receptor EFR to the intracellular domain of the phylogenetically related rice receptor Xa21 yielded a functional chimeric receptor in both rice and Arabidopsis Holton et al. This receptor triggers defense markers when transgenic tissues are treated with elf18, the ligand of EFR, although whole-plant resistance to the microbe was weak or undetectable Holton et al. Conversely, expression of a fusion receptor consisting of the ectodomain of Xa21 and the cytoplasmic domain of EFR in rice conferred robust resistance to Xoo expressing the cognate ligand of Xa21 Thomas et al.

Two related studies reported that chimeric receptors generated by fusing the rice chitin-binding protein Chitin Elicitor-Binding Protein and the intracellular protein kinase domain of the rice receptor-like kinase Xa21 or Pi-d2 confers disease resistance to the rice blast fungus Kishimoto et al. Although these studies have not been advanced to field trials, they demonstrate that domain swapping among immune receptors may be an attractive approach in engineering broadened recognition specificity.

NLRs often perceive pathogens indirectly by monitoring the modification of host target proteins by pathogen-derived virulence factors Jones and Dangl, Some of these host target proteins have evolved to serve as decoys that are targeted by virulence factors van der Hoorn and Kamoun, Defense is triggered when the plant decoy protein PBS1, a kinase, is cleaved by the protease AvrPphB secreted by the bacterial pathogen Pseudomonas syringae into the plant cell Ade et al.

Kim et al. For example, they found that they could remove the cleavage site of PBS1 that is recognized by the AvrPphB protease and replace it with cleavage site variants recognized by other pathogen proteases such as the AvrRpt2 protease from Pseudomonas syringae , or the Nla protease from Turnip mosaic virus Kim et al. The engineered forms of PBS1 are cleaved in vivo by these proteases, which activates RPS5 defense in response to the corresponding pathogen strains Kim et al.

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We previously demonstrated that a DNase gene transferred to a plant along with a proper promoter is capable of inducing the total defense response in the recipient plant against that plants true pathogens. The Impact: there is an unlimited source of these DNases that when manipulated properly may transfer stable disease resistance to any tranformable plant species. Thus we discovered an unlimited source of disease resistance genes for plants.

Publications Hadwiger L. These DNases are of importance since they have the potential to activate a response in plant tissue that can protect the plant from pathogens with the potential to infect plant tissue. DNases were detected and or purified from 5 strains of Verticillium dahliae known as pathogens of potato, spinach, mint, sugarbeet, strawberry and watermelon.

Also from Puccinia striiformis v. The mode of action of the DNase is inducing individual PR pathogenesis-related genes. The gene transcription initiating action occurred as a result of chromatin changes, by ubiquitination of nuclear proteins and cleavage of DNA strands both of which appeared to allow the transcription complex to proceed along the open reading frame of the defense gene.

Impacts During the initial sequencing of fungal DNases and the designation of mitochondrial DNases by cross-referencing with the BLAST program it was discovered that all of the fungi that have had a total sequence reported have a related DNase gene and most of these can be excreted from the fungal cell.

These DNase activities all appear to have the potential to activate the total defense response in the pea tissue that enables it to resist pea pathogens.

Thus this signal-inducing ability when transfered to plant tissue transgenically can serve as an authentic disease resistance gene. The fungal DNases therefore serve as an unlimited source for disease resistance in plants.

Publications No publications reported this period. Fungal releases of DNase are capable of activating the entire immune response non-host resistance response in peas against the true pathogen, F. We have shown that these DNases can exit the fungus, enter the plant cell, and cause moderate damage to the host plant's DNA. As a result the RNA polymerase complex is then able to move from the gene's promoter region through the open reading frame of the defense gene PR gene. Regions of the chromosome, identified by other researchers mapping quantitative loci QTLs , often including open reading frames of PR genes we have cloned, appear to be more sensitive to these alterations.

Information acquired in this non-host resistance system demonstrates how immunity may develop in many types of plant disease resistance. For future research I will continue to sequence, clone and further characterize DNases released by the additional genera and follow the corresponding ubiquitination and PR gene induction.

Impacts The concentration of my research on elicitors such as DNase is to provide the basic knowledge that will support the practical aspects of crop development. For example we have previously used a gene promoter in combination with a DNase gene in a construction that was transferred to both tobacco and potatoes with resulting increases in disease resistance. That is, when it is known what genes actually carry out resistance and how they are regulated, then new resistance traits can actually be created.

Further, the cloning of the DNases from a number of genera can provide major insights into how the most stable form of plant disease resistance, non-host resistance, functions to protect a given plant from the many plant pathogens in its environment.

Cloning the plant disease resistance genes activated provides valuable markers for regions of the chromosome for those mapping and trying to locate regions of chromosomes for transfer in conventional breeding schemes.

Our research has also benefited breeders. The innovation of my laboratory is its focus on alterations at the nucleosome level that are responsible for the activation of Pathogenesis-Related PR genes. The understanding of two of the mechanisms involved is now essentially complete and is as follows: The pathogenic fungus releases DNase via an inherent, cleaved N-terminal sequence that facilitates transfer through membranes, enters the plant cell and moves to the nucleus causing single strand nicks in the DNA of chromatin.

Subsequent chromatin loosening allows the increased transcription of PR genes in sensitive regions [defined by investigators of quantitive traits QTLs ]. Also released is the fungal cell wall compound, chitosan, that also enters the plant nucleus. Chitosan has a high affinity for DNA and is capable of altering chromatin structure again activating PR genes. Both of these signals promote a total disease resistance response against a true pea pathogen. The chromatin alteration is associated the diminution of histones H2A and H2B, proposed by others to enable the RNA polymerase complex to progress through and thus transcribe the open reading frame to protein.

Currently I have purified DNases from five major classes of plant pathogen fungi and determined they also induce disease resistance. Impacts This research has provided a molecular explanation for how plants respond to signals to activate PR defense genes genes and generate a total resistance response in peas. It has identified the components that can be used to develop through genetic engineering, total disease resistance in all transformable plant species. The gene for the external signal, DNase, has been cloned and can now be transcribed internally within the plant an its induction controlled by an inducible promoter from a PR gene promoter.

Publications Hadwiger, L. Hadwiger, L. A complete text book-like documentation of the processes and components that develop within 6 h of the peas' total immunity non-host resistance to Fusarium solani f. The F. The latter elicitors are released and enter the plant nucleus. The premature internal accumulation of FsphDNase in F. Additional analysis of the migration and localization of many gene products involved in host-parasite interactions has been assembled via computerized-prediction programs based on their N-terminal sequences.

These changes occurring at the nucleosome level have been shown to be central to transcriptional increases in many eucaryotic systems, but except for our research, this area of specialization in plant disease resistance has been overlooked.

Impacts The field of plant host-parasite interactions has been dominated in recent years by those performing mutational genetics on Arabidopsis to indirectly predict the biochemical effects and signaling occurring in these interactions. The impact anticipated for this work, which combines all of the actual biochemical effects as well as the actions of purified signals in a highly research model, is to show the actual components that activate the plant defense response as well as those that cause the suppression of growth of the challenging fungus.

The total "non-host" defense response in pea endocarp tissue to Fusarium solani f. All cellular proteins are normally recycled by the ubiquitination process, however in the pea defense response this process is highly accelerated.

As a result of eliminating or temporarily dislocating HMG A or histones from the chromatin structure in the vicinity of the genes appears to assist the progression of the polymerase complex to and through the open reading frame. I am currently utilizing a chromatin immuno precipitation technique which has enabled me to determine that these nuclear protein changes occur in the vicinity of our PR genes at their chromatin location. In a cooperative development, ARS researcher Dr.

Clare Coyne, has discovered that several of these defense genes co-map with quantitative traits QTLs in peas. Different conc. Therefore I propose that the information will be very valuable to other plant pathologists when they also pursue this direction. I have just initiated studies using a technique called chromatin immunoprecipitation which will allow me to know if the alteration of these nuclear proteins occurs in the realm of the individual PR gene sites on chromatin.

Impacts We cloned the major pea PR defense genes in the s and early 90s. Though their potential was recognized it was not fully recognized until their contribution to plant immunity was tested. Individual genes first bolstered resistance in potato and tobacco because pea transformations were difficult; we showed that individual genes could enhance the recipient plant's ability to resist certain diseases.

Clare Coyne has recently shown that the qualitative traits she has identified through mapping, co-map with several of the pea genes we cloned. A major potential is now attainable knowing gene functions, map locations and DNA sequences of traits that reasonably can assemble and form immunity on the level of the non-host resistance we see in peas against bean pathogens.

Publications Hartney, S. The use of chemical genomics to detect functional systems affecting the non-host disease resistance of pea to Fusarium solarni f. Thus the modifications of HMG A appear to be vital to non-host resistance in pea plants. This architectural TF is key in the chromatin modeling that controls transcription of certain genes. Both the accumulation and the phosphorylation of HMG A decrease in direct association with the activation of PR genes.

Theoretically the absence of or phosphorylation of this TF enables the polymerase complex a more advantageous site to initiate the transcription of the gene.

In agreement with this concept, extremely low levels of the phosphatase inhibitor, calyculin A, applied to pea totally induces a resistance response to pea pathogens. Thus, this pea system enabled a definition of the pathogen initiated gene activating signal route all the way to chromatin modeling within the plant nucleus, an analysis not reported from other plant defense systems.

Progress continues with the practical applications of the natural polymer, chitosan. This natural polymer serves as a sticker in combination with copper containing materials. Chitosan improves the efficacy of copper sulfate: as a fungicide, an aquatic herbicide, and as an eliminator of fairy ring spot a fungal-based invasion of lawn grasses.

These advances have been particularly important to organic potato growers that depend on natural ingredients such as copper sulfate to combat potato late blight. The 40 fold increase in efficacy enables disease control without the residual copper accumulations that can accumulate following the necessary repetitive applications normally required to suppress the disease. Impacts The plant science paper covering the research of inhibitors that block vital plant cell processes, is available at no cost on line, and I hope that the identification of vital biochemical processes that are involved with the non-host resistance of peas to plant pathogens will be of interest to those who currently depend on mutational analysis.

The mutational information and biochemical investigations may eventually arrive at similar conclusions. However at present I suggest that the roles of specific proteins and the states of phosphorylation can be most directly pursued at the biochemical level, knowing the DNA sequences that are targeted in non-host resistance and the major roles played by nuclear protein modifications.

Plant Science The published role of chitosan in distributing and stabilizing the action of copper based fungicides and herbicides should encourage others to investigate its efficacy in other plant systems. Plant Health Progress doi The use of chemical genomics to detect functional systems affecting the non-host diease resistance o pea to Fusarium solani f.

Pisum Genetics Low-level copper plus chitosan applications provide protection against late blight of potato. Plant Health Progress. In non-host resistance" the incompatible pathogen, Fusarium solani f. These inhibitors induce copious quantities of pisatin when applied singly and enhance the induction synergistically when applied with chitosan, a natural elicitor released by Fusarium. The elicitors, salicylic acid, jasmonic acid, super oxide and nitric oxide generators were ineffective in inducing resistance in this legume system.

Chitosan in addition to inducing resistance in peas, has physical properties useful in attaching fungicides, herbicides or algaecides to plant and algae targets to control late blight of potato, submerged invasive weeds and algae. Impacts Impact statement: The physical sticker properties of chitosan have the potential for organic growers to use in the control of potato late blight when combined with copper sulfate pentahydrate. Both this disease control and the weed control when combined with sand and herbicide show efficacy with only a fraction of the recommended pesticide application rate.

This promoter, driving a gene for DNase from Fusarium solani f. The treatment did exceed copper oxide also approved for organic growers at much lower concentrations. A procedure was developed for surveying pathogen-induced gene signaling routes, called the "inhibitor array technique". Using all available inhibitors of vital cellular processes, those involved with the non-host resistance of pea against the bean pathogen, F. The processes influenced included pisatin production, hypersensitivity, PR gene activation, fungal growth, DNA damage, cell death and resistance.

Impacts The processes studied have a central role in the understanding and development of disease resistance in plants. The spin off benefits will be to growers, environmentalists and recreational enthusiasts. The chitosan-related treatments have already benefited the organic potato grower and the control of invasive weeds will render benefits in navigation, water drainage systems and fish stocks. In the last year we have completed a major analysis of the promoter from the model Pathogenesis-related gene DRR from peas.

The promoter functions in at least three plant species. In another strategy for plant protection,we have devised a control for potato late blight. Last year the product was supplied to 4 grower-cooperators and we are in the process of legalizing the treatment for the organic grower.

Impacts The promoter study discribed above has been used in a genetic engineering scheme for disease resistance driving the DNase gene we have reported on previously. This construction has successfully increased the disease resistance in two plant species.

The control treatment described above involving chitosan and copper sulfate is one of the few prospects for protecting potatoes grown by the organic farmer. The mode of action of chitosan and its oligomers in inducing plant promoters and developing disease resistance in plants.