Team:Wageningen UR/Results/Plant Immune Response

Xylencer

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Triggering a Plant Immune Response

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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.

Introduction

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.

Results

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.

Figure 1: The lesion size of leaves of Brassica oleracea on day 3, 4 and 5 after inoculation with flg22, compared to a control (H2O).
Healthy plant inoculated with flg22. There are no visible results. This image was taken in a specialized climate room.
Lesion development on a leaf. A small lesion can be seen at the edge of the cut mark on the leaf. Faint yellowing of the leaf is visible there. At this point the leafs were inoculated with flg22.
Advanced lesion on a leaf. Yellowing of the edge of the leaf is visible, as well as blackening of the veins. This image was taken in a specialized climate room.

Conclusion

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.

  • General Approach arrow_downward

    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).

  • References arrow_downward
    1. Jones, J. D., & Dangl, J. L. (2006). The plant immune system. nature, 444(7117), 323.
    2. Choi, H. W., & Klessig, D. F. (2016). DAMPs, MAMPs, and NAMPs in plant innate immunity. BMC plant biology, 16(1), 232.
    3. Zipfel, C., Robatzek, S., Navarro, L., Oakeley, E. J., Jones, J. D., Felix, G., & Boller, T. (2004). Bacterial disease resistance in Arabidopsis through flagellin perception. Nature, 428(6984), 764.
    4. Newman, K. L., Almeida, R. P., Purcell, A. H., & Lindow, S. E. (2003). Use of a green fluorescent strain for analysis of Xylella fastidiosa colonization of Vitis vinifera. Appl. Environ. Microbiol., 69(12), 7319-7327.
    5. Rapicavoli, J. N., Blanco-Ulate, B., Muszyński, A., Figueroa-Balderas, R., Morales-Cruz, A., Azadi, P., ... & Roper, M. C. (2018). Lipopolysaccharide O-antigen delays plant innate immune recognition of Xylella fastidiosa. Nature communications, 9(1), 390.
    6. Baccari, C., Antonova, E., & Lindow, S. (2018). Biological control of pierce’s disease of grape by an endophytic bacterium. Phytopathology, 109(2), 248-256.
    7. Felix, G., Duran, J. D., Volko, S., & Boller, T. (1999). Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. The Plant Journal, 18(3), 265-276.