Cyst nematodes are economically important plant pathogens that induce vast subcellular
changes in host root cells to form a specialised feeding site, the syncytium. Previously
characterised changes to the plant secretory pathway during syncytial formation include
the proliferation of the endoplasmic reticulum (ER) and Golgi apparatus, and the
replacement of the large central vacuole with numerous smaller vacuoles. To further
characterise the plant secretory pathway in plant-cyst nematode interactions, novel dual
fluorescence marker constructs were developed. Each construct contained a Golgi
marker fused to YFP and an additional plant secretory pathway marker fused to RFP.
Stable Arabidopsis marker lines expressing these constructs were infected with the beet
cyst nematode Heterodera schachtii to provide a model host-cyst nematode system.
Fluorescence microscopy evidenced small vacuoles throughout the syncytium as
expected. However, all other plant secretory markers within the dual fluorescence lines
were undetectable in syncytia, suggesting disruption to the plant secretory pathway. To
support this, gene expression analysis of a subset of plant secretory pathway genes was
conducted using published RNA-seq data. Results from this suggest the altered
regulation of genes involved in the early secretory pathway and post-Golgi trafficking,
validating the fluorescence microscopy observations.
Another aim of this work was to identify novel cyst nematode effectors containing a single
C-terminal transmembrane domain (TMD), that are predicted to localise to the ER. For
this, a multi-step bioinformatics pipeline was created, using the proteomes of Heterodera
schachtii and the potato cyst nematode Globodera pallida. Eight screened nematode
cDNAs were cloned, with tobacco leaf epidermal cells transformed to analyse subcellular
localisation, and in-situ hybridisations conducted to validate gland cell expression
indicative of effector activity. From this, five novel putative effector genes were identified,
localising to varied subcellular compartments, including the ER, nucleus, and
peroxisomes. This effector screen has contributed to growing evidence that plant
pathogen effectors can have transmembrane domains, and if studied further, these
genes could provide cyst nematode target genes for RNA interference.
