This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
Sweet potato (Ipomoea batatas) is an extremely important staple food crop, particularly in Africa. It serves as a vital source of carbohydrates, along with a variety of different minerals and vitamins. Root-knot nematodes (RKNs), obligate parasites of the roots of many plant species, pose a significant threat to sweet potato production. To facilitate parasitism, RKNs secrete effectors to manipulate plant immune responses and promote growth. Some of these effectors can be detected by intracellular immune receptors known as nucleotide-binding domain leucine-rich repeat-containing proteins (NLRs). NLR-mediated immunity is usually accompanied by the hypersensitive response, a form of localised cell death that restricts pathogen spread.
In Asterids, NLRs form complex networks comprised of sensors, that are involved in the detection of pathogens, and a conserved set of helpers known as NLRs-required for cell death (NRCs), which mediate downstream immune responses. While this has been extensively studied in Solanaceous plants, the NLR network remains underexplored in the Convolvulaceae family, which includes sweet potato. The NRC superclade, encompassing both NRCs and sensors, has expanded in Ipomoea species, making it more challenging to characterise compared to the NRC network in plants belonging to the Solanaceae family. Despite these challenges, understanding the NRC network in sweet potato is essential, as it represents the majority of the plant’s NLRome and has direct implications for resistance-breeding strategies. Therefore, the aim of this project was to elucidate functional NRCs and uncover sensor-helper NLR combinations within sweet potato.
Before the start of my internship, the sweet potato genome was scanned and annotated for NLRs, and a phylogenetic tree of the NLRome was generated. To identify NLRs for cloning, I selected NRCs and sensors across clades to better represent a broader portion of the NLRome. Additionally, NLRs that were differentially expressed in sweet potato roots infected with the root-knot nematode Meloidogyne incognita were also cloned. These NLRs were PCR-amplified and cloned into E. coli using Gibson cloning.
After verifying the plasmids via Nanopore sequencing, site-directed mutagenesis was performed to make the NLRs constitutively active, enabling their functional testing without requiring pathogen effectors to trigger an immune response. A single amino acid substitution, usually aspartic acid to valine, within a well-conserved NLR motif known as the MHD leads to NLR autoactivity. This autoactivity arises from increased ATP binding in an NLR domain, leading to the adoption of an active protein conformation. It is important to note that sensors require their corresponding NRCs to trigger downstream immune responses. Therefore, functional testing of sensor NLRs necessitates co-infiltration with wild type NRCs. Whilst NRCs are not involved in effector perception, they can be mutated to become autoactive, allowing their functionality to be assessed independently.
Agrobacterium tumefaciens was transformed with binary vectors carrying the constitutively active NLRs using electroporation. The function of the NLRs were assessed using Agrobacterium-mediated transient gene expression in the model organism Nicotiana benthamiana. Functional NRCs were expected to trigger a hypersensitive response upon constitutive activation, observable as cell death in N. benthamiana leaves. Given that the corresponding NRCs are lacking in N. benthamiana, the NRC-dependent sensor NLRs from sweet potato would induce cell death if co-infiltrated with the specific NRCs they signal through in sweet potato.
During my internship, I made substantial progress in cloning various NLRs. One newly cloned NRC and seven previously cloned NRCs were successfully tested for function. In addition, I tested the function of one NRC-dependent sensor with these eight NRCs, and it appeared to signal through three of them. By the end of my internship, four additional NRCs and three sensors were in the final stages of cloning and prepared for transformation into Agrobacterium. Additionally, five NRCs and five NRC-dependent sensors were ready to be made constitutively active. Following on from my internship, this project will be continued through identifying and cloning more NLRs, with the aim of unravelling the NRC network in sweet potato and investigating the role of the NLRs in RKN resistance.
This summer studentship was an invaluable insight into scientific research and plant pathology. I was able to hone my lab skills and learn many new techniques such as Gibson cloning and agroinfiltration. I thoroughly enjoyed my time at the Crop Science Centre and would like to thank everyone in the Derevnina lab, in particular Unnati Sonawala for all the support and help on the project. Finally, I would like to thank the BSPP for this opportunity.
Ella Greensmith
University of Cambridge
Cover image: Cell death assay using agrobacterium-mediated transient expression of wild-type and constitutively active NRCs from sweet potato in Nicotiana benthamiana leaves. N. benthamiana leaves were infiltrated with Agrobacterium tumefaciens carrying plasmids for expression of wild-type sweet potato NRCs on the left and the corresponding constitutively active form on the right. Cell death resulting from the expression of the constitutively active form of the NRCs can be visualised by the fluorescence UV light. This fluorescence is a result of the accumulation of phenolic compounds during cell death. Leaves were imaged under UV light at 5 days post infiltration. The red background of the leaves is a result of the autofluorescence from chlorophyll pigments in the leaves.
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
