This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
Plant parasitic nematodes (PPN) are a devastating crop pest, causing significant crop losses globally. There are over 4,100 species of PPN, including Globodera pallida (potato cyst nematode, PCN) and Meloidogyne incognita (root-knot nematode, RKN). PCN infects solanaceous plants and is the most damaging potato pest in the UK, while RKN infects over 3,000 plant species, including major crops. Nematode virulence is controlled by factors including nematode effector molecules, resistance or tolerance of host, and interacting biotic factors such as arbuscular mycorrhizal fungi (AMF).
Upon hatching from their eggs, second-stage juvenile (J2) nematodes penetrate roots, developing through J3 and J4 stages to mature females by feeding through an induced feeding site within the root. As the nematode only begins feeding when in a suitable location within the root, the J2 only has access to its innate and finite energy supply before root penetration. Recent lab work highlighted the potential importance of Sugar Will Eventually be ExporTed (SWEET) genes in potatoes during PCN infection therefore, I explored the potential role of these genes in RKN infection.
In-situ hybridisation (ISH) shows gene expression within nematodes by staining the RNA transcript of a gene. ISH was used to locate SWEET4 expression in RKN. First, a probe complementary to SWEET4 transcripts was synthesised. A ~150 base pair region of SWEET4 gene cDNA was amplified, and the product purified. This ~150 base pair purified DNA was used in a PCR cycle using the reverse SWEET4 primer and digoxigenin-labeled nucleotides (DIG-dNTPs) to create a single-stranded complementary probe. DIG-dNTPs are heavier than normal nucleotides, yet this probe appeared lighter when run on a gel against the original PCR product, indicating the probe may not have been successfully synthesised. Probe synthesis was repeated, adjusting annealing temperature and concentration of PCR product to increase DIG-dNTP incorporation. Again, this was not successful, so the entire process was repeated using a different region of the SWEET4 gene.
Once the SWEET4 probe was created, J2 RKNs (the mobile stage that moves through soil to find a root) were prepared, fixed to preserve RNA, cutting to allow the probe to enter, and permeabilising. The SWEET4 probe was hybridised with the nematodes, and then incubated with anti-DIG antibody complementary to the DIG-dNTPs used in the probe. The nematodes were then stained to allow the anti-DIG antibody to be visualised with brightfield microscopy. Staining, and therefore SWEET4 expression, was often in the gut area (Figure 1). SWEET expression in J2s indicates sugar uptake from the gut, which is interesting as J2s have no access to plant sugars. This indicates that SWEETs may be involved in the movement of sugars derived from the breakdown of nematode lipid reserves, pre-invasion.
Figure 1: J2 Meloidogyne incognita (Root Knot Nematode). Left, unstained worm; right, worm hybridised with SWEET4 probes, appearing as a brown-purple colour in the worm.
To explore the potential impact of biotic interactions on nematode parasitism, sections of AMF-colonised root and sections of AMF+PCN female-infected root from 4 different potato cultivars of varying PCN tolerance were cut and frozen (Cover Image). The aim was to understand how root-AMF-PCN interactions relate to plant tolerance of PCN, as previous work had shown that AMF may provide tolerance against the parasite. RNA sequencing of these root sections may reveal how the tripartite interactions vary across potato cultivars at a molecular level and if there are any limits to tolerance. This information could be useful for breeding PCN-tolerant potatoes.
Unexpectedly, previous research had shown potatoes infected with AMF had more RKN than those without AMF. To check if the same phenomenon occurred with tomatoes (a potato relative), the number and life stage of RKN on tomato plants with and without AMF was recorded. Acid fuchsin highlighted the worms within the tissue, allowing RKN to be counted in each root section. Results showed tomatoes had fewer RKN with AMF, than tomatoes without AMF, i.e. the opposite trend to what was observed in potato plants. Comparing root gall RNA transcripts from tomatoes and potatoes may uncover gene expression differences that confer AMF-induced immunity to RKN in tomato.
The work I did with the Plant Nematology group at the University of Leeds introduced me to a new area of plant pathology and allowed me to learn various molecular and nematode-specific techniques. This helped guide my future career towards research. Thanks to everyone in the Plant Nematology group, particularly Chris Bell and Willow Maxwell, for their support during the placement.
Cover image: Potato root containing female Globodera pallida (PCN).
Alex Papp
Cornwall College
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
