This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
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The yellow potato cyst nematode, Globodera rostochiensis, is a sedentary endoparasitic pathogen that infects solanaceous plants, such as potatoes and tomatoes. It enters the roots of plants initially as motile juveniles (J2), and then establishes permanent feeding sites (J3). Cysts are then formed on the roots, containing hundreds of eggs that can remain dormant for over 20 years, making the nematodes extremely hard to eradicate. In the UK, potato cyst nematodes are estimated to cost the potato industry £50 million annually and can result in tuber yield losses that exceed 80%. Despite the economic importance of this pathogen, little is known about the molecular mechanisms by which G. rostochiensis causes disease in plants. Nonetheless, like other plant pathogens, plant parasitic nematodes secrete proteins known as effectors to facilitate infection. Uncovering how these effectors enable successful invasion, is crucial to develop our understanding of parasitism. Therefore, our aim was to identify effectors in G. rostochiensis and investigate their functions in plants.
In cyst nematodes, effectors are predominantly produced and secreted from oesophageal glands, known as the dorsal and subventral gland cells. Therefore, we can classify genes encoding secreted proteins as effectors if they are highly expressed in gland cells. As the gland cells of G. rostochiensis have not been sequenced yet, a closely related cyst nematode species with complete gland cell libraries, Heterodera schachtii, was used to identify homologous effectors in G. rostochiensis.
Firstly, I identified all the effectors in the model cyst nematode H. schachtii. Using gland cell sequencing data, categories of gene expression were created, and hypergeometric tests were performed per category using two parameters: the number of secreted proteins and the number of known effectors. In general, previously known effectors were all shown to be highly expressed in the library. This means that previously uncharacterised genes in those same expression categories or higher, provided they also encode putatively secreted proteins (presence of N-terminal signal peptides, and the absence of transmembrane domains or endoplasmic reticulum retention signals), are likely also effectors. Therefore, the lowest expression category with significant enrichment in effectors (p-value <0.01) was chosen as a threshold (Figure 1) and those genes predicted to encode secreted proteins in that category or above were designated as putative effectors. For example, if the expression of a gene was greater than 256 in the gland cells of J2 nematodes, it was deemed a putative effector if it encodes a protein that is predicted to be secreted. The procedure was repeated for the J3 gland cell library, settling on a threshold of 2038. With these thresholds, 479 effectors were predicted for H. schachtii.
With a comprehensive and high confidence list of effectors collated, 506 orthologues of H. schachtii effectors were identified in G. rostochiensis. To determine which orthologues were secreted and thus likely corresponding and conserved effectors, the G. rostochiensis deduced amino acid sequences were queried for N-terminal signal peptides, and the absence of transmembrane domains or endoplasmic reticulum retention signals. This resulted in 233 of the orthologues being classed as effectors in G. rostochiensis, of which 64% were putative.
To clone the effectors, I obtained the cDNA of J2 nematodes 4 days after inoculation (Figure 2). Then I designed primers with golden gate overhangs for 54 putative G. rostochiensis effectors that were expressed in the parasitic stages, and out of these I cloned 23 effectors successfully and assembled them into plasmids. These plasmids were then transformed into E. coli, verified with colony PCR, and sequenced. The correctly sequenced clone was transformed into Agrobacterium tumefaciens.
To test the function of the cloned effectors, I agroinfiltrated them into the leaves of the model Solanaceae plant, Nicotiana benthamiana. Each effector was expressed individually to determine whether they induced an immune response in the host. A total of 6 replicates were carried out. None of the effectors were shown to induce an immune response in the leaves of N. benthamiana.
Most cloned plant disease resistance genes encode for nucleotide-binding domain and leucinerich repeat-containing (NLR) proteins, which are involved in activating immune responses. In Solanaceae plants, NLRs form networks, comprised of sensor NLRs, involved in detecting pathogen effectors, and a conserved set of helper NLRs known as NRCs (NLR-required for cell death) which translate effector recognition into hypersensitive cell death. Previous work revealed that some cyst nematode effectors target NRCs I, therefore, hypothesised that the putative effectors of G. rostochiensis may also suppress NRCs. To test this, I co-infiltrated the putative effectors with autoactive forms of NRC3 (NRC3D480V) or NRC4 (NRC4D478V) and assessed the cell death phenotype at 5 days post infection (Figure 3). I tested a total of 12 of the 23 effectors. I included an empty vector as a negative control and SS15, a G. rostochiensis effector that suppressed NRC3D480V but not NRC4D478V mediated cell death, as a positive control. None of the 12 effectors I tested suppressed either NRC3 or NRC4 mediated cell death. However, these effectors may suppress other NRCs (e.g. NRC2), which will need to be tested. In addition, the function of the other 11 cloned effectors will also need to be tested. Ultimately, by identifying and characterising host targets of effectors, we can then better engineer crops for resistance against potato cyst nematodes.
This studentship has been a great insight into plant pathology. I have had the opportunity to develop my Python and R, and learn many new laboratory skills such as RNA extraction, golden gate cloning, and agroinfiltration. These skills will be invaluable in any future research career I pursue. I would like to thank all the researchers in the SEVDA lab at the Crop Science Centre, and my supervisors Lida Derevnina and Sebastian Eves-van den Akker for all their support and advice throughout my project.
Mariam Ahmad
Imperial College London
Figure 1. Determining the minimum gene expression in gland cells needed to classify a secreted protein as a putative effector in Heterodera schachtii. The minimum gene expression decided for each gland cell library is highlighted in green.
Figure 2. Collecting G. rostochiensis cDNA during the parasitic stages.
Figure 3. N. benthamiana leaves showing hypersensitive response after co-expression of G. rostochiensis effectors with empty vector (negative control), SS15 (positive control), and autoimmune NRC3D480V and NRC4D478V mutants. Results from four effectors shown. Plants were photographed 5 days after agroinfiltration.
