Xylencer aims to make use of two killing mechanisms. To completely eradicate X. fastidiosa we
aim to
trigger an immune response in the plant besides phage therapy. Xylem inoculation with the flg22 peptide,
a Microbe Associated Molecular Pattern (MAMP) that is recognized by the plant immune system, temporarily
reduces disease symptoms in plants. flg22 shows a positive effect after the appearance of visible
lesions and can therefore be applied as a treatment. Healthy plants inoculated with flg22 did not show
any visible symptoms.
Unlike mammals, plants cannot depend on an adaptive immune system or mobile immune cells for protection
against pathogens. To protect themselves against diseases, plants rely on the innate immune system of
individual cells and signaling molecules sent by infected cells [1]. Specifically, the innate immune
system of plants consists of two branches. The first branch is the recognition of Microbe- (or
Pathogen-) Associated Molecular Patterns (MAMPs/PAMPs) [1]. MAMPs are conserved molecules present in
diverse kingdoms, including molecules such as bacterial flagellin (flg) and lipopolysaccharides (LPS)
[2]. These MAMPs are recognized by Pattern Recognition Receptors, leading to PAMP triggered Immunity
(PTI) and thus temporary resistance against pathogens in plants [3].
X. fastidiosa can spread aggressively throughout the xylem and remain there for years [4]. It
manages to
thrive in the xylem as it camouflages the part of its LPS that is recognized by the receptors with a
long O-antigen chain, thereby delaying the onset of PTI. When purified LPS, with the immunogenic regions
exposed, is inoculated in the xylem a PTI response is triggered. This response is strong enough to
temporarily prevent infection by X. fastidiosa [5]. Intriguingly, if a X. fastidiosa
infected plant is
inoculated with an endophyte that triggers an immune response within four weeks of introduction of
X.
fastidiosa, the population size of the pathogen reduces to almost zero [6].
In this research, we aimed to find the effect of xylem inoculation of MAMPs on vascular pathogens. We
tested the effect of flg22, a peptide consisting of 22 conserved amino acids at the N-terminus flagellin
of Pseudomonas syringae, on disease resistance when inoculated in the xylem [7]. Flg22
is a well described MAMP and has been shown to trigger disease resistance [3].
This effect was tested on a pair of model organisms in plant pathology. Xanthomonas campestris
pv.
Campestris (Xcc) is a vascular pathogen but, unlike X. fastidiosa, is not considered as a
quarantine
organism, eliminating safety concerns. Brassica oleracea (cabbage) is a suitable host plant for
Xcc
infections, as it shows clear symptoms and xylem inoculation is relatively easy due to the leaf
structure and size. Plants were infected with Xcc and were xylem-inoculated with flg22 upon the
appearance of lesions. The difference in lesion size between leaves receiving flg22 treatment and a
control was determined to quantify the effect of flg22 treatment.
To test the effect of flg22 on disease progression, it was xylem-inoculated in Brassica oleracea that
showed symptoms of disease.
The lesion size in leaves xylem inoculated with flg22 was significantly smaller compared to the negative
control three days after treatment (p < 0.05). This difference in lesion size diminished the
following two
days. At day 4 and day 5, there was no significant difference in lesion size between the flg22
treatment and the negative control. This indicates that the effect of flg22 treatment is temporary.
There was no visible reaction in healthy plants that were xylem inoculated with flg22. Treatment with flg22 is successful in diminishing disease symptoms in Brassica oleracea infected with
Xcc. This positive effect is visible when flg22 is applied after the appearance of disease symptoms.
This indicates that flg22 is not only suitable for priming plants immune defenses before infection with
a pathogen, as described in literature, but can also be used as a treatment [3]. The
positive effect of treatment with flg22 is temporary. The effect is gone within 4 days. With Xylencer,
flg22 will be supplied as long as Xylencer phages infect X. fastidiosa. flg22 might be
successful in
treating X. fastidiosa infected trees.
The plant
protocols were used in this research.
This research was performed with Brassica Oleracea Goldon Acre and Xcc LMG 568.
Six infected plants were xylem inoculated for each condition. Any attempts to purify recombinantly expressed MAMPs was unsuccessful. More information on
our troubles
with expressing proteins can be found on the Protein library page.
Purification attempts
were performed with flg22 (BBa_K3286138) and NLP20 (BBa_K3286139). Triggering a Plant Immune Response
Introduction
Results
Conclusion