Abstract:
Cercospora zeina is the causal agent of grey leaf spot (GLS) of maize, which causes
devastating crop yield losses. The fungus secretes molecules termed effectors to overcome
host defences, obtain nutrients from the host and negatively affect the host. In this study, two
effectors in C. zeina and their orthologs from Cercospora zeae-maydis were identified as good
candidates to study based on in silico analyses. This included analyses by EffectorP 3.0,
Localizer 1.0, ApoplastP 1.0 SignalP 6.0. The two candidate effectors in C. zeina were named
CzCSEP4 and CzCSEP20, and in C. zeae-maydis termed CzmCSEP4 (89% identity to
CzCSEP4, on the protein level) and CzmCSEP20 (94% identity to CzCSEP20, on the protein
level). These genes were expected to cause a hypersensitive response in Nicotiana
benthamiana in the form of cell death. The genes were amplified from C. zeina or from C.
zeae-maydis cDNA and their native signal peptides were replaced with an apoplastic-specific
signal peptide. These effectors were then cloned into a binary vector system, transformed into
Agrobacteria, and infiltrated into N. benthamiana. In N. benthamiana the positive control of
INF1 (Phytophthora infestans) showed necrosis, whereas the two C. zeina candidate effectors
did not (p>0.05, t-test). The effectors were demonstrated to have structural similarities to the
Fulvia fulva Ecp2 effector’s Hce2 (Homologs of C. fulvum Ecp2) domain. Effectors containing
this domain were able to elicit a hypersensitive response in several tobacco species, including
N. tabacum, and could not elicit a response in N. benthamiana. These genes were then tested
in N. tabacum and showed a hypersensitive response in the infiltrated areas that was
significantly different to the Agrobacterium only control (p<0.05, t-test). In conclusion, these
genes can be termed secreted effector proteins, thereby proving that these genes are likely to
form part of an effector gene family and may play a role in promoting pathogenicity of the fungi
in the host.
