Physalis peruviana, known as cape gooseberry, is a solanaceous
plant native to tropical South America, typically growing in the
Andes at 2000 m. Its economic value has grown due to its
nutritional and medicinal properties. However, a vascular wilt
disease caused by a newly discovered forma specialis of Fusarium
oxysporum, (here designated f. sp. physali [Foph]), has become
one of the limiting factors in cape gooseberry production, with
losses up to 90%.
The F. oxysporum species complex incudes numerous formae
speciales (special forms), which are the causal agents of
vascular wilt disease in a broad range of plants, including
economically important crops such as banana, cotton, melon,
tomato and recently, cape gooseberry. F. oxysporum f. sp.
lycopersici (Fol) causes wilt disease on tomato. At least
fourteen small secreted in xylem (SIX) proteins have been
identified from the xylem sap of Fol-infected tomato plants. Five
are associated with virulence and three are recognised by
resistance proteins in the host. However, the function of most of
these SIX proteins remains unclear.
In this project, six homologues of Fol SIX genes (SIX1a, SIX1b,
SIX7, SIX10, SIX12 and SIX15) and a homologue of Ave1 (an
avirulence gene present in the broad-host-range wilt-pathogen
Verticillium dahliae, with homologues in many other
phytopathogens including Fol), were identified in Foph. These and
other candidate effector genes were identified by mapping Foph
RNAseq data against the Fol lineage-specific transcriptome and
candidate F. oxysporum effector genes identified in other formae
speciales in other studies. The Foph SIX gene and Ave1 homologues
were found to encode proteins with 70 to 100% identity to their
Fol counterparts, with the latter suggesting recent horizontal
transfer of a cluster of SIX genes comprising SIX7, SIX10, SIX12
and SIX15.
The Foph SIX1a and SIX1b proteins are 74% and 80% identical,
respectively, to their Fol counterpart. Both homologues were
tested in a ΔSIX1 strain of Fol to see if they could complement
the virulence function of Fol SIX1 in tomato. The results showed
no restoration of virulence for ten SIX1a and six SIX1b
transformants tested, suggesting that their function might be
restricted to cape gooseberry pathogenicity. Foph SIX1a and SIX1b
transformants were also tested to see if they might be recognised
by tomato plants carrying the I-3 resistance gene, which enables
recognition of Fol SIX1 (i.e. Avr3). The results indicated that
SIX1a was not recognised while SIX1b was recognised, suggesting
that Foph-SIX1b may be recognised by I-3 as an avirulence factor
and that the I-3 resistance gene could potentially be used in
cape gooseberry plants to mediate Foph resistance.
VI
To investigate the function of SIX7, SIX10 and SIX12, a triple
gene knockout strategy was initiated to assess their role in Fol
virulence. This strategy included the use of the HSVtk (Herpes
Simplex Virus thymidine kinase) gene as a counter selection
marker against the ectopic insertion of transfer DNA (T-DNA)
during fungal transformation by Agrobacterium tumefaciens.
However, after several transformation attempts no gene knockouts
were obtained. Attempts to produce single (SIX10) and double
(SIX7 and SIX12) knockouts also failed.
The Fol Rapid Alkalinisation Factor (RALF) gene was also
subjected to gene disruption using this approach. Four RALF
knockouts were obtained out of 44 transformants thereby
validating the gene deletion strategy described above.
Pathogenicity tests in tomato showed that these four mutants all
developed disease symptoms that were not significantly different
from those of wild type Fol under the assay conditions used
