Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803
Performing Department
Plant Science
Non Technical Summary
The potato cyst nematodes,Globodera pallidaandGlobodera rostochiensis, have caused substantial financial losses to potato growers throughout the temperate zones.In the UK, where up to 9% of the potato crop has been lost to these pests, this has amounted to£50 million per year, but in other parts of the world such as areas of Spain and Russia where harvests have been reduced by as much as 80%, the effects have been even more economically devastating.In 2006, an area of 8,478 acres spread over two counties in southern Idaho recorded outbreaks ofG. pallida.A great deal of work is currently underway to identify naturally resistant potato cultivars and to breed the best of those resistances into commercially important linages such as Russet Burbank that are favored by the Idaho potato industry.It is unclear how long this work will take, and how long the resistance will persist until a nematode mutation allows it to be evaded.However, there are some species that developed a long-lasting form of nematode resistance.One such example isSolanum sisymbriifolium(SSI), commonly called litchi tomato or sticky nightshade.This non-host, like sensitive crops such as potato, secretes chemicals that "tricks" otherwise dormant nematode eggs into hatching.The J2 larvae then chemotax towards SSI roots, invade, but are then killed or otherwise prevented from undergoing further molts to sexual maturity.In an effort to determine how this is accomplished, we have performed experiments that have identified a set of genes potentially participating in a nematode-resistant defense.The purpose of the present study isfirst and foremost,to identify genes in SSI that protect it from cyst nematodes, andsecond,to determine if these genes are capable of providing novel forms of nematode resistance in transgenic crops such as potato to hedge against future infestations.This research will expand the currently small repertoire of genes available for enhancing plant resistance to not onlyG. pallidaandG. rostochiensis, but other plant parasitic nematodes as well.
Animal Health Component
(N/A)
Research Effort Categories
Basic
95%
Applied
(N/A)
Developmental
5%
Goals / Objectives
Solanum sisymbriifolium(SSI), commonly called litchi tomato or sticky nightshadeIs resistant to a parasitic nematode of potatoes called Globodera pallida.This plantt secretes chemicals that "trick" otherwise dormant nematode eggs into hatching in the absence of a suitable host.The J2 larvae then chemotax towards the trap crop roots, invade, but are then killed or otherwise prevented from undergoing further molts to sexual maturity. The purpose of this study isto identify genes in SSI that protect it fromnematodes, anddetermine if these genes are capable of providing novel forms of nematode resistance in transgenic crops such as potato. We have identified 277 genes that change expression when SSI roots are infected by G. pallida. We have selected several of these genes for further study. Goal 1 will be to isolate and sequence the selected genes, and introduce them under the control of a strong promoter, into potatoes. The resulting plants will be then evaluated for increased nematode resistance. Objective 1:Isolate and sequence 4 kinase- and receptor kinase-genes from SSI, 2 NDR1/HIN1-like genes from SSI, and at least 4 representative peroxidase genes from SSI.Objective 2:Clone genes into plant expression vectors and transform them into potatoes.Objective 3:Assess the resistance of the transformed potatoes againstG. pallida, and additional pathogens such asPseudomonas syringaepv. DC3000,Phytophthora infestans, andMeloidogyne incognita, as facilities allow. Goal 2will be to develop effective protocols for stably transforming SSI so that we can knock out the selected genes and assess whether their loss renders SSI sensitive. Objective 4 will be to develop a protocol for SSI transformation using abeta-glucuronidase carrying T-DNA vector. If successful, we will then use CRISPR-Cas9 or RNAi to reduce expression of the genes under study in Objective 1. In this way, we will demonstrate whether these genes are essential for nematode resistance.
Project Methods
ForObjective 1. Using RNA-seq, we identified 277 nematode-induced genes.From these, we chose 3 sets of genes for further study.Set 1genes are putative receptors that might activate defenses in response to nematode invasion.Our analyses identified 6 differentially-expressed kinases and receptor kinases.Set 2will focus onndr1/hin1-likeproteins which have previously been shown to enhance resistance to both bacterial and fungal pathogens(Chen, Tian, et al., 2018).We identified 2 versions of this gene in our transcriptome.One was induced by nematodes, and one, with 64% identity to the induced gene, was not.Set 3genes are the most autonomously-acting of the 3 sets.This set consists of peroxidase genes.The 3 most common roles for these proteins are to contribute to host cell death, damage the pathogen's surface, and polymerize lignins and extensin polymers. We have selected 4 for further study.Each gene candidate will be amplified from primary cDNA prepared from RNA taken from the roots of nematode-infected SSI following standard protocols using Phusion DNA polymerase (New England Biolabs, Ipswich, MA) and cloned into a TOPO-TA Gateway vector (ThermoFisher Scientific, Waltham, MA), according to manufacturer's recommendations.Each will be sequencedusing Sanger protocols.Evaluation:Since our SSI reference library database had been reduced to a CD-HIT-EST(Li and Godzik, 2006)of 90, cloning will be judged successful if each isolated cDNA isat least90% identical to the gene in our SSI database.Efforts:Successful clones and sequences may be used in classroom demonstrations of sequencing technologies.ForObjective 2:Sanger-verified sequences will be recombined from the pENTR vectors into the pEARLY100 T-DNA-based vector(Earley, Haag, et al., 2006)for transformation intoAgrobacterium tumefaciens, and then introduced into Desiree potatoes.Transformants will be selected on phosphinothricin (PPT)-containing shoot-inducing medium.Evaluation:Plants will be judged transformed if they 1) test as sterile when grown repeatedly on medium without bactericidal agents, 2) test as transformed if they can produce root hairs from roots growing into medium containing sufficient PPT to kill untransformed roots, and 3) contain the new gene when assayed by PCR and gene-specific primers.Efforts:Successful plants will be photographed and subsequently incorporated in tests whose results will be used in future publications, and shown to students in classroom demonstrations.ForObjective 3:We will preserve a minimum of 5 transgenic plants harboring each constructfor further serial propagation in sterile medium.Once these 5 lines have become established, at least 3 plants derived from each line will be transferred to soil in isolated greenhouses, and challenged withG. pallida.While our primary aim is to developG. pallida-resistant plants, we will later challenge our plants withP. syringaepv DC3000, and possibly withPhytophthora infestansandMeloidogyne incognita, as facilities allow, to assess whether any of our genes could have alternative or additional benefits.We will conduct semi-quantitative RT-PCR to estimate the expression of each transgene relative to the SSI actin gene,Ssi032526 (Wixom et al., submitted), in order to determine whether the level of resistance for any of these pathogens correlates with the expression of the introduced receptor.Should any one of the peroxidases decrease susceptibility toG. pallida, we will endeavor to define its operation by correlating the resistance of independent transgenics with the activity of the introduced enzyme(Dowd, Lagrimini, et al., 1998, Kawaoka, Matsunaga, et al., 2003), and, since many of the SSI proteins have cell-secretion sequences, by building new constructs fused to an antigenic tag(Earley, Haag, et al., 2006)so that we can determine whether the peroxidases accumulate intra- or extra-cellularly.We will also remove the secretion sequences from any extracellular candidates, or add N-terminal signal peptides, to see if enhanced nematode resistance is lost.Evaluation:We will measure the effect that the transgene has on cyst number and the number of viable eggs/cysts after nematode infection.A collaborator (L-M Dandurand, U. of Idaho) has been carrying out these very tests on WT potatoes and SSI for several years(Hajihassani and Dandurand, 2018, Whitworth, Novy, et al., 2018),We will then use RT-PCR to identify which organs express the original gene in SSI and quantify how this expression changes during pathogen infection.Efforts:Any gene found to alter pathogen resistance will be studied in depth and be the subject of at least one scientific publication.Any student working on this project will be strongly encouraged to present their progress in the form of posters at University and commodity research meetings.ForObjective 4: We attempted to establish a protocol forAgrobacteriuminfection of SSI so that we could introduce constructs to make those modifications.We found, however, that SSI resistedAgrobacteriumas effectively as it resisted nematodes. One of the major points made in the resulting transient expression paper(Wixom, Casavant, et al., 2018)was that SSI showed a form of age-related resistance(Hu and Yang, 2019).In or work, age correlated with the number of leaves on each plant: there were significantly more GUS+spots on plants with 8-14 leaves than on plants with 25-31 leaves. Given these observations, we now intend to concentrate on regenerating plants from even "less mature" material: the shoot apex and hypocotyls of SSI seedlings.SSI seeds have been obtained from both field-grown and greenhouse-grown plants (J. Kuhl, University of Idaho).After sterilization, seeds are placed on water agar (no salts or other nutrients) petri dishes in the dark. Over a period of 4-8 weeks, we have seen approximately 10% of these seeds germinate, with some variation according to the batch of seeds.Pre-treating them with 10mM gibberellic acid (GA3) for 24 h increased the germination frequency to 80%, but only in some batches of seeds.In order to develop a reliable transformation protocol, we will pre-induce theAgrobacteriumstrain, GV3101 (pGV2260) carrying the vector pCAMBIA1301 with acetosyringone(Wixom, Casavant, et al., 2018).We will then add approximately 105bacteria to petri dishes containing etiolated SSI seedlings that had been germinated with and without 10mM GA3.Special care will be taken to minimize damaging of the seedlings at this stage to avoid triggering the extreme wound reaction observed on larger plants(Wixom, Casavant, et al., 2018).After allowing the bacteria 24 h at 28oto infect, seedlings will be placed in ½ strength Murashige and Skoog medium supplemented with 300 mg L-1timentin.After 3 d, these seedlings will be cut into 2-3 pieces and placed on agar containing the same salts and antibiotics together with 10 mg L-1hygromycin, 2.5 mg L-1trans-zeatin, and 0.1 mg L-1IAA.The petri dishes will be incubated in the dark.Dying segments of the seedlings will be excised as needed, while proliferating calli or shoots will be put in 18 h light.Shoots that form recognizable leaves will be transferred to the same medium without hormones so that they can form roots.Evaluation:Hygromycin-resistant shoots will be transferred to½ strength MS with 300 mg L-1timentin so that they can form roots.These plants will be propagated sterilely in medium without timentin.We will then verify first, that they are sterile, second, that they can form root hairs in medium with hygromycin, and finally, they are transformed by performing GUS assays, and PCR analyses.If this succeeds, we will introduce RNAi or CRISPR-Cas9 constructs todetermine if inactivatingchosen genes renders plants nematode-sensitive.Efforts: The story of how we produced GUS-positive plants despite the many obstacles would be a valuable lesson for both college students and outside visitors in how science sometimes has to proceed. ?