Histopathological Aspects of Induced Resistance by Pseudomonas Protegens CHA0 and Β-Aminobutyric Acid in Wheat Against Puccinia
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bioRxiv preprint doi: https://doi.org/10.1101/2020.02.06.934943; this version posted February 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Histopathological aspects of induced resistance by Pseudomonas protegens CHA0 and β- 2 aminobutyric acid in wheat against Puccinia triticina 3 Fares BELLAMECHE1, Mohammed A. JASIM1*, Brigitte MAUCH-MANI1, Fabio MASCHER 2§ 4 1 Laboratory of Molecular and Cell Biology, University of Neuchâtel, Switzerland 5 2 Plant Breeding and Genetic Resources, Agroscope Changins, CH-1260 Nyon, Switzerland 6 * Present address: Biology department, University of Anbar, Al Ramadi 31001, Iraq 7 § For correspondence: [email protected] 8 Abstract 9 After perception of specific biotic or abiotic stimuli, such as root colonization by rhizobacteria or 10 selected chemicals, plants are able to enhance their basal resistance against pathogens. Due to its 11 sustainability, such induced resistance is highly valuable for disease management in agriculture. Here 12 we study an example of resistance against wheat-leaf rust induced by Pseudomonas protegens CHA0 13 (CHA0) and β-aminobutyric acid (BABA), respectively. Seed dressing with CHA0 reduced the 14 number of sporulating pustules on the leaves and the expression of resistance was visible as necrotic 15 or chlorotic flecks. Moreover, a beneficial effect of CHA0 on growth was observed in wheat 16 seedlings challenged or not with leaf rust. BABA was tested at 10, 15 and 20 mM and a dose- 17 dependent reduction of leaf rust infection was observed with the highest level of protection at 20 mM. 18 However, BABA treatment repressed plant growth at 20 mM. Balancing between BABA-impact on 19 plant growth and its protective capacity, we selected 15 mM as suitable concentration to protect 20 wheat seedlings against leaf rust with the least impact on vegetative growth. To understand the 21 mechanisms behind the observed resistance, we have studied the histological aspects of the fungal 22 infection process. Our results showed that the pre-entry process was not affected by the two 23 resistance inducers. However, both treatments reduced fungal penetration and haustoria formation. 24 The timing and the amplitude of the resistance reactions was different after bacterial or chemical 25 induction, leading to different levels of resistance to leaf rust. During fungal colonization of the 26 tissues, a high deposition of callose and the accumulation of H2O2 in both CHA0- and BABA-treated 27 plants pointed to an important contribution to resistance. 28 Key words: 29 Leaf rust, callose deposition, hydrogen peroxide (H2O2), plant resistance inducers 30 INTRODUCTION 31 Plants dispose of several layers of sophisticated defense mechanisms to defend themselves against 32 pathogen attack. The first layer is given by preformed physical and chemical barriers that impede the 33 pathogen to penetrate into the plant and to initiate infection (Ferreira et al. 2006). Once the presence 34 of the pathogen has been detected, the plant activates further chemical and physical barriers that 35 block or at least delay the attack (second layer; Jones and Dangl (2006)). Defense success depends 36 on the readiness of the plant to detect the pathogen. In the case of the interaction between wheat and 37 the leaf rust pathogen (Puccinia triticina), the plant can detect specific fungal avirulence factors 38 (elicitors) with leaf rust resistance genes (Lr). This gene-by-gene interaction is a very rapid 39 recognition-reaction event leading to an elevated degree of resistance against the disease. However, 40 the avirulence patterns can change and the pathogen may become undetectable by the plant. This 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.06.934943; this version posted February 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 41 resistance breakdown happened recently with yellow rust (Hovmøller et al. 2010) and stem rust 42 (Singh et al. 2011). 43 In the case of unspecific recognition of the pathogen, the plant is still able to contain the 44 development of the pathogen but with a reduced and variable degree of severity of infection (Jones 45 and Dangl 2006). The degree of this quantitative resistance is linked to the readiness of the plant 46 defenses and depends on a series of genetic and environmental factors. Besides the pathogen itself, 47 biological and abiotic stimuli as well as certain chemicals can enhance plant resistance (Mauch-Mani 48 et al. 2017). Such induced resistance can be limited to the site of the inducing treatment but it can 49 also be systemic and thereby effective in parts of the plant distant from the site of induction (Van 50 Loon 1997). For instance, certain root-associated bacteria such as the biocontrol strain Pseudomonas 51 protegens CHA0 (formerly P. fluorescens CHA0) induce systemic resistance against viral and fungal 52 diseases in various dicots (Maurhofer et al. 1994; Haas and Keel 2003; Iavicoli et al. 2003) and 53 monocots (Sari et al. 2008; Henkes et al. 2011). Certain chemical compounds can also induce 54 disease resistance in plants, e.g. the non-protein amino-acid β-amino-n-butyric acid (BABA). Root 55 colonizing bacteria and BABA root treatment reduce significantly the severity of infection caused by 56 the oomycete Hyaloperonospora arabidopsis on Arabidopsis thaliana, and the induced state is 57 regulated by different defense signaling pathways, depending on the inducing agent and the 58 challenging pathogen (Van der Ent et al. 2009). 59 In the present work, we aimed to study the mechanisms underlying induced resistance by CHA0 and 60 BABA in wheat against leaf rust. A previous study has shown that root colonization by 61 Pseudomonas protegens strain CHA0 reduces the number of leaf rust uredia on susceptible wheat 62 seedlings in wheat (Sharifi-Tehrani et al. 2009). The enhanced resistance is very likely due to a 63 resistance priming event by induction of systemic resistance (ISR). This priming enables the plant to 64 cope with the pathogen at an early stage of infection. To study this, in the work presented here, we 65 followed the interaction between the plant and the pathogen at the microscopic level (De 66 Vleesschauwer et al. 2008). 67 The infection process of leaf rust is well known (Bolton et al. 2008). After adhesion of a 68 urediniospore on the leaf surface, germination, directed growth of the germ tube on the plant surface 69 towards a stoma, and recognition of the guard cell lips take place. A small appressorium is formed 70 over the stomatal opening and then, a penetration hypha is entering through the stomatal pore. 71 Following penetration, a substomatal vesicle, and haustorium develop (Bolton et al. 2008). 72 Primed plants recognize the pathogen and produce reactive oxygen species (ROS) and deposit 73 callose at the infection sites (Balmer et al. 2015). This rapid local oxydative burst generates, among 74 other, hydrogen peroxide (H2O2) during pre-haustorial resistance against wheat leaf rust caused by P. 75 triticina (Wesp-Guterres et al. 2013; Serfling et al. 2016). Callose is an effective barrier that is 76 induced at the sites of attack during the early stages of pathogen invasion (Luna et al. 2011). A 77 strong deposition of callose has been reported for the wheat Thatcher near-isogenic lines carrying 78 leaf rust resistance genes (Wang et al. 2013). 79 Few studies have investigated rhizobacteria- and BABA-induced resistance against wheat leaf rust. 80 In this study, we aimed to compare mechanisms involved CHA0-ISR and BABA-IR during 81 interaction between leaf rust and wheat. To this end, we evaluated microscopically the development 82 of fungal structures, the occurrence of callose deposition and hydrogen peroxide accumulation in leaf 83 tissues. 84 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.06.934943; this version posted February 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 85 MATERIALS AND METHODS 86 Induced resistance assay 87 Plant material and growth conditions 88 Experiments were done with the leaf rust-susceptible bread wheat cultivar Arina (Agroscope/DSP). 89 Surface sterilized seeds were used in all experiments. Wheat seeds were rinsed with 70% ethanol, 90 incubated for 5 minutes in 5 % bleach (sodium hypochlorite solution, Fisher Chemical, U.K.) and 91 washed three times in sterile distilled water. The sterilized seeds were germinated on humid filter 92 paper (Filterkrepp Papier braun, E. Weber & Cie AG, 8157 Dielsdorf, Switzerland) in plastic bags 93 maintained in the dark at room temperature. Three to 4 days later, seedlings at similar growth state 94 and morphology were selected and planted in 120 mL polypropylene tubes (Semadeni, 3072 95 Ostermundingen, Switzerland) filled with a standard potting mixture (peat/sand, 3:1, vol/vol). The 96 tubes were placed in a growth chamber with the 16 hours day at 22°C and 8 hours night at 18°C and 97 with 300 mol m-2 s-1 light. The plants were watered regularly keeping the potting soil wet yet 98 avoiding its saturation. 99 Bacterial inoculum 100 The bacterial inoculum consisted of the biocontrol agent P. protegens strain CHA0-Rif (Natsch et al.