Effects of exogenous salicylic acid and pH on pathogenicity of biotrophy-associated secreted protein 1 (BAS1)-overexpressing strain, Magnaporthe oryzae
Abiotic stress can influence the interactions between a pathogen and its host. In this paper, we analyzed the effects of salicylic acid (SA) and pH on the morphological development and pathogenicity of Magnaporthe oryzae, the pathogen that causes rice (Oryza sativa) blast. A strain of rice blast that overexpresses biotrophy-associated secreted protein 1 (BAS1) and a wild-type (WT) strain were pretreated with different levels of pH and different concentrations of SA to analyze M. oryzae colony growth, sporulation, spore germination, dry weight of hypha, and appressorium formation. Disease incidence and the expression of defense-related genes in infected rice were analyzed after pretreatment with pH 5.00 or pH 8.00 and 200 μM SA. The results showed that both SA and pH had some influence on morphological development, including sporulation and appressorium formation of the BAS1-overexpression strain. In the 200 μM SA pretreatment, there was a lower incidence of disease and higher expression levels of the rice defense-related genes PR1a, PAL, HSP90, and PR5 on leaves inoculated with the BAS1-overexpession strain compared with the WT strain, whereas, LOX2 appeared to be downregulated in the BAS1-overexpession strain compared with the WT. In both pH treatments, disease incidence and expression of HSP90 were higher and the expression of PR1a and PR10a and LOX2 and PAL was lower in leaves inoculated with the BAS1-overexpression strain compared with leaves inoculated with the WT strain. We conclude that SA and pH affect morphological development of the BAS1-overexpression blast strain, but that these factors have little influence on the pathogenicity of the strain, indicating that BAS1-overexpression may have enhanced the tolerance of this rice blast strain to abiotic stressors. This work suggests new molecular mechanisms that exogenous SA and pH affect the interactions between M. oryzae and rice.
KeywordsMagnaporthe oryzae Rice Salicylic acid pH Pathogenesis-related genes Abiotic stress
Allene oxide synthase 2
Biotrophy-associated secreted protein 1
Crinkling and necrosis induced protein
Enhanced disease susceptibility 1
Heat shock protein 90
Programmed cell death
Potato dextrose agar
Potato dextrose broth
Phytophthora sojae crinkling and necrosis induced protein 115
Quantitative real-time polymerase chain reaction
Phenylalanine ammonia lyase
The effector proteins secreted by plant pathogens play a key role in the interaction between the pathogens and the host plant as well as in the progression of the disease. Effector proteins are considered to manipulate the host’s cell structure and function, facilitate infection, and suppress the host’s immune response (Kamoun 2006; Hogenhout et al. 2009; Cooper et al. 2016). Once effector proteins enter the host, they can both work in the extracellular matrix and change the host’s cellular environment to facilitate infection and colonization (Kamoun 2006; Ridout et al. 2006; Hogenhout et al. 2009; Białas et al. 2017).
Pathogens secrete various enzymes based on their surrounding environment (Gebauer et al. 2017; Gumtow et al. 2017; Kaverinathan et al. 2017). During the infection period of Botryis cinerea, the fungus can secrete various intracellular polygalacturonases to facilitate its infection (Louis et al. 2014). Environmental conditions may also play a role; many studies have reported that temperature and pH can influence the secretion of effector proteins by pathogens. Louis et al. (2014) found different soluble candidate effector proteins were secreted by Cochliobolus lunatus at different temperature conditions. Some fungi have evolved complicated regulatory mechanisms to recognize and respond to the surrounding pH changes, such as secreting different proteins according to different pH levels in the surrounding environment (Li et al. 2012). To invade its host successfully, B. cinerea can secrete different proteins in different tissues of the host (Sharma et al. 2016). Although some real virulence factors can successfully suppress a host’s defense response, they still need to function in coordination with specific extracellular secreted proteins (Li et al. 2012). The study on B. cinerea shows that the culture solution of B. cinerea is black under the conditions of pH 4 and pH 6 because of the secondary metabolites produced during the growth of the hyphae (Sharma et al. 2016; Kaverinathan et al. 2017). A separate study showed that the microenvironment (pH) of the host plant could regulate an arsenal of enzymes to increase fungal pathogenicity (Alkan et al. 2013). Thus, we can conclude that pH is a major environmental factor that affects the fungus secreting effector proteins and promotes its colonization in the host’s tissues. Other studies have shown that the C. lunatus isolates with higher melanin levels are more virulent than those with lower melanin levels; thus, melanin and related secondary metabolite are considered to be virulence factors of C. lunatus (Xu et al. 2007; Gao et al. 2012).
M. oryzae (Ascomycotina) cause the fungal disease rice blast, which has serious effects on rice (Oryza sativa) production, severely impairing rice yield and quality. Under normal conditions, conidia that fall on the surface of the host plant start to germinate. Germ tubes quickly differentiate to form specialized infected cells, i.e., the appressoria. M. oryzae have a highly specialized infection structure, an appressorium, which penetrates the host plant (Howard et al. 1991; Jong et al. 1997). After penetrating the host, the invasive hyphae grow rapidly in the host cells and, concurrently, spindle-shape lesions appear. After 5 to 7 days, a large number of conidia are produced on lesions and a new round of the infection cycle begins. During infection, the microenvironment of the host plant cells can change, including the pH, temperature, and humidity around the infection site. The host cells may also increase or decrease levels of hormones, such as salicylic acid (SA) and jasmonate acid (JA), which can affect signal transduction and the production of lysates and secondary metabolites. For example, the expression of the PR1 gene can be induced by SA and JA (Mitsuhara et al. 2008).
There are also many reports about how the signal transduction pathway in fungal cells is related to pH level. Fungi can grow and develop in a wide range of pH levels because fungi can regulate their surrounding pH by secreting acidic or alkaline substances (Piccirillo et al. 2010; Pen et al. 2011; Landraud et al. 2013); these secretions can further facilitate its ability to infect and colonize a host plant. Thus, environmental factors (e.g., temperature, humidity, pH, active oxygen, and SA) are predicted to play an important role in the interaction between the M. oryzae and its rice host. Therefore, understanding the effects of the environmental factors on the interaction between the M. oryzae and the host could help to reveal new pathogenic mechanisms of M. oryzae.
This paper studied the effects of an exogenous hormone (SA) and pH on the sporulation, spore germination, appressorium formation, and mycelial growth, pathogenicity of a strain of M. oryzae that overexpresses biotrophy-associated secreted protein 1 (BAS1). This study aims to provide a platform for further analyses of the effect of abiotic stress on the interaction between the pathogen and its host plant, and provide important theoretical and experimental proofs for guiding the agricultural management.
Materials and methods
The wild-type (WT) strain of M. oryzae (95234I-1b) used here is stored in our laboratory. Strain 35S:BAS1/Mo-2 is a transformed strain that overexpresses BAS1; it is also stored in our laboratory and was obtained by transforming the expression vectors of BAS1 and mCherry fusion under the 35S promoter constructed.
Lijiangxintuanheigu (LTH) was used in this paper; the rice variety was a universally susceptible variety, which is also stored in our laboratory.
Culture of blast strain in media containing different concentrations of SA and different pH values
Mycelial pellets of rice blast strain were placed on a PDA (potato dextrose agar) medium, and then put in a 28 °C incubator for 7 days. The fresh mycelia pellets in PDA were put into a triangular flask filled with potato dextrose broth (PDB) culture medium, and then put the triangular flask in a shaker of 28 °C and 120 rpm for 3 days of culture. We dissolved the SA (Sigma, MO, USA) in a small amount of dimethyl sulfoxide (DMSO) and prepared the culture media for the different SA concentrations. The control (CK) treatment consisted only of the quantity of DMSO required for preparing the highest SA concentration and an aliquot of sterile water. The six SA treatments were as follows: 50 μM, 100 μM, 200 μM, 500 μM, 1 mM, and 2 mM. The pH was adjusted for each culture media (PDA, PDB), by adding 20 mmol/L MES buffer solution or 20 mmol/L MOPS buffer solution and then further adjusted using NaOH or HCl (Landraud et al. 2013).
Effects of SA concentration and pH on the growth of M. oryzae mycelium
We used mycelia pellets with a puncher of 7 mm in diameter and inoculated them onto PDA media containing different SA concentrations or different pH values. Pellets were placed in the 28 °C incubator for culture. We measured the colony diameters on day 10.
Spore production of M. oryzae
We inoculated 300 μL M. oryzae culture solution into Ximeizhi media containing different SA concentrations and different pH levels and then placed them in a 28 °C incubator for culture for 4 days in the dark, followed by alternate light and darkness (12:12, dark:light) for 6 days. We washed the cultured M. oryzae spores with 5 mL sterile water and filtered them with gauze. The filtered solution was mixed uniformly and the spores were counted with a blood-counting chamber.
Observation of spore germination and appressorium formation in M. oryzae
We adjusted the sterile water containing buffer solution into an aqueous solution of different pH values with NaOH and HCl. We collected the spores of M. oryzae from Ximeizhi media. SA solution was added to adjust the spore concentration to 1 × 105 spores/mL. We then took 10 μL of the spore suspension and added it to a hydrophobic slide, and placed the slide in a 28 °C constant-temperature petri dish. We observed spore germination after 2 h and appressorium formation after 6 h.
Culture and inoculation of rice seedlings
Rice seeds were sterilized for about 1 min in hypochlorite before sowing. Seeds of LTH were soaked in sterilized water and placed into a 28 °C constant-temperature incubator until dehiscent, and then sown into a seedling bed. Rice seedlings were used for the inoculation experiments when they had formed three leaves (approximately 20 days). To prepare the M. oryzae inoculum, we washed the M. oryzae spores with sterile water, centrifuged them for about 1 min and discarded the supernatant. Next, we added sterile aqueous solutions of pH 5.00, pH 8.00, or CK (sterile water), and SA solutions of 200 μM with CK (DMSO solution required in 200 μM SA), to adjust the spore suspension to a concentration of 1 × 105 spores/mL, and added in 0.02% Tween 20. The inoculant was applied as a spray to the entire seedlings. Seedlings were kept in the inoculation hood for 24 h after inoculation to preserve the heat and humidity in the darkness after which they were transferred to the greenhouse. About 15 leaves were taken for quantitative analysis 0, 24, 48, 72, 96, and 120 h after the spray inoculation. Seven days after inoculation, we performed a disease investigation. Disease incidence rate was calculated as the number of infected leaves divided by the total number of leaves.
Total RNA extraction, cDNA reverse transcription, and qRT-PCR
Primer sequences of the target genes in rice leaves
Primer pair (5′ → 3′)
Data analysis method
For the qRT-PCR data processing and statistical analysis, we used the 2−ΔΔCt method (Livak and Schmittgen 2001). Three biological replicates and three technical replicates were included in all experiments testing the effects of SA and pH on the growth of mycelium, sporulation quantity, spore germination and appressorium formation, disease investigation, and qRT-PCR. All data and statistical analyses were performed in SPSS 13.0. A two-way analysis of variance (ANOVA) was carried out, followed by the Duncan’s multiple range tests. Graphs were plotted with Sigmaplot 10.0.
Effects of SA concentration and pH level on the morphological development of blast strains
To identify the effects of different concentrations of SA and different pH values on the morphological development of the BAS1-overexpression strain, we treated plants inoculated with different concentrations of SA and different pH values (BAS1-overexpression strain and a WT strain of 95234I-1b treated with the same DMSO or sterilized water as controls).
Colony growth of the BAS1-overexpression strain was lower in the pH 5.00 medium than in the control; however, the difference was not significant. BAS1-overexpression strain cultured in pH 8.00 medium had a significantly higher colony growth than that of the control (Fig. 1). Overall, pH highly affects the BAS1-overexpression strain comparing to the WT strain.
Effects of different concentrations of SA and different pH values on the pathogenicity of blast strains
We next made an analysis of the pathogenicity of BAS1-overexpression isolates under different concentrations of SA and pH. BAS1-overexpression and WT strains were pretreated with 200 μM SA and pH of 5.00 or 8.00, and disease symptoms were investigated 7 days after the inoculation.
Disease incidence of spores on rice leaves pretreated with salicylic acid (SA) and inoculated with wild-type (WT) or BAS1-overexpression rice blast strains (Magnaporthe oryzae). Values show the means ± SD of three biological replicates. Different letters represent significant difference at P ≤ 0.05
Disease incidence (%)
47.37 ± 4.71b
55.3 ± 5.46a
200 μM SA
32.07 ± 2.03c
41.83 ± 0.85b
Disease incidence in rice leaves pretreated with pH 5.00 or pH 8.00 and inoculated with a wild-type strain (WT) or BAS1-overexpression strain (35S:BAS1/Mo-2) of rice blast. Values show the means ± SD of three biological replicates. Different letters represent significant difference at P ≤ 0.05
Disease incidence (%)
36.6 ± 5.7b
45.67 ± 2.84a
43.93 ± 2.29a
46.97 ± 1.32a
Effects of SA and pH on defense system of rice infected by blast strains
The expression level of LOX2 in leaves inoculated with the BAS1-overexpression and WT strains pretreated with 200 μM SA appeared downregulated during infection, but there was more downregulation in leaves inoculated with the BAS1-overexpression strain than in leaves inoculated with the WT strain (Fig. 7). The expression level of AOS2 in leaves inoculated with BAS1-overexpression strain pretreated with 200 μM SA appeared a little upregulation during infection comparing with from 24 to 72 hpi, and it appeared higher expression level of AOS2 in leaves inoculated with the BAS1-overexpression strain than in leaves inoculated with the WT strain at 96 and 120 hpi (Fig. 7).
Expression of LOX2 in leaves inoculated with the BAS1-overexpression strain and the WT strain pretreated with pH 5.00 and pH 8.00 was significantly downregulated during infection, and lower regulation was observed in leaves inoculated with the BAS1-overexpression strain pretreated with both pH values. The expression quantity of AOS2 in rice seedlings inoculated by spores of overexpression isolates treated under pH 5.00 and pH 8.00 was significantly upregulated during infection (Fig. 10).
Climate change could create the environmental conditions for a disease triangle (host plant, pathogen, and environmental conditions suitable for disease development) and this could intensify the occurrence of epidemic plant diseases (Mcelrone et al. 2007). Changes in environment conditions can have a direct impact on pathogens and host plants. During the interaction between M. oryzae and rice, M. oryzae secretes effector proteins to regulate and manage the cellular structure and physiological metabolism process of rice before infecting the host.
By analyzing the effects of different pH values on the morphological development of blast strains, we found that different pH values had certain effects on the colony growth, spore germination, and other aspects of blast strains; however, it appeared there was no significant effect on the colony growth of WT strains. Landraud et al. (2013) found that the M. oryzae strain Guy11 cultured in PDA (potato dextrose agar) medium with pH between 5.00 and 8.00 experienced no significant differences in colony growth. Trushina et al. (2013) also found that the deletion mutant ΔpacC of the Trichoderma transcription factor PacC grew at a slow rate in alkaline PDA. Effector proteins have certain acid–base properties, and they can change the acid–base properties of their microenvironment after secreting the effectors, which can promote or suppress the growth of the pathogens. The effect of different pH levels on mycelia growth of several species of dematiaceous fungi (Chai and Liu 2010) and the influence of low pH on arbuscule development, and inhibition effect has been shown to increase (Feng et al. 2017).
We also found that different pH values had an influence on the morphological development of the BAS1-overexpression strain, and that different pH levels could increase the disease incidence rate in rice infected by overexpression isolates. Zou et al. (2010) reported that the function of a few cells is controlled by pH, but the function of these cells was related to pathogen-infecting plants. In this paper, we found some genes appeared upregulated and others downregulated. The expression level of the pathogenesis-related genes PR1a and PR10a decreased in leaves inoculated with the overexpression strain pretreated with the two pH values. It is well known that the PR1a gene is related to the PCD (programmed cell death) signal pathway, and its high expression level promotes the death of a large number of cells in an infected rice seedling and thus suppresses further infection by pathogens (Wu et al. 2014; Fekih et al. 2015). However, in this study, the expression level of PR1a decreased, indicating that the death of the rice cells was suppressed, and this facilitated further infection of the overexpression strain. Concurrently, we found that the expression level of LOX2 and the PAL also decreased, indicating that the JA and SA signal pathways of the infected rice seedlings were suppressed, i.e., the defense response of the infected rice seedlings was also suppressed.
Heat shock protein 90 (HSP90) is one of the most broadly studied proteins in HSP families. It functions in cells as a molecular chaperone in response to stress conditions (Yan et al. 2017). We found that the expression level of HSP90 was upregulated at 24 hpi in leaves inoculated with the overexpression strain and, since HSP90 is a stress response gene, the upregulated expression of this gene indicated that the infected rice seedlings were under stress response instead of defense response. In the infected rice seedlings, the upregulation of HSP90 together with the decrease of expression level of PR1a promoted the infection and colonization of the overexpression strain.
SA is a metabolite produced in the plant infected by the pathogens by itself and is bound to affect the morphological development of the pathogens to different degrees. SA could induce enzymatic antioxidant activities, related gene expression such as PR1a and PR10a (Xie et al. 2011; Gill et al. 2016; Kanno et al. 2012), and heat shock protein 90 (HSP90) was one of the most broadly studied proteins in HSP families. It functioned in cells as molecular chaperones in response to stress conditions (Yan et al. 2017). Pathogenesis-related proteins-1 (PR-1), glucanase (Glu), and chitinase (Chi) genes are widely considered is a defensive gene for SA-dependent signaling pathways (Glazebrook 1999).
We found that different concentrations of SA had certain effects on the morphological development of the BAS1-overexpression strain, and we also found a significant decrease in disease incidence rate, and the expression level of PR1a and PR10a in leaves infected by rice blast strain pretreated with 200 μM SA. These effects appeared to be greatest at the early stages of infection and gradually decrease in the later stages of infection, indicating that SA induces an early defense response in infected rice plants and that the death of rice cells was suppressed at the late stage of infection. PR1a is a gene related to the PCD signal pathway, and its expression promotes the death of a large number of cells in an infected rice seedling and thus inhibits further infection of the pathogens (Wu et al. 2014; Fekih et al. 2015). SA plays a key role in the signal transduction path of plant response to biotic stress (Horváth et al. 2007), and the expression of the stress response gene HSP90 in leaves infected by the overexpression isolates pretreated with 200 μM SA was upregulated at 24 h, indicating that the infected rice seedlings were under significant stress response at the time. In addition, we found that the expression of the PAL gene gradually increased over time, indicating that exogenous SA induced the SA signal pathway of the infected rice seedlings. So the BAS1-overexpression strain had higher tolerance to abiotic stress than WT strain. Zhang et al. (2015) found that the expression of the CRN effector protein PsCRN115 of Phytophthora sojae in the Nicotiana benthamiana improved the resistance of N. benthamiana to biotic stress and abiotic stress. Therefore, it can be seen that the expression of pathogen effector proteins in either plants or the pathogens helps improve their tolerance to the external biotic stress and abiotic stress.
JY, YW, LL, LL, and CW carried out the experiment work. JY and CL helped in drafting the paper and interpreting the data. All authors read and approved the final manuscript.
This work was supported by the National Key R&D Program of China (2017YFD0200400), the National Natural Science Foundation of China (Grant No. 31400073), and the Yunnan Natural Science foundation (Grant No. 2013FB039) from the Yunnan Science and Technology Department of China.
- Białas A, Zess EK, De JLC, Franceschetti M, Pennington HG, Yoshida K et al (2017) Lessons in effector and nlr biology of plant-microbe systems. Mol Plant Microbe Interact. MPMI08170196FIGoogle Scholar
- Chai B, Liu WD (2010) Effection of different pH on the growth of several species of Dematiaceous fungi. J Trop Med 10:815–816Google Scholar
- Fekih R, Tamiru M, Kanzaki H, Abe A, Yoshida K, Kanzaki E, Saitoh H, Takagi H, Natsume S, Undan JR, Undan J, Terauchi R (2015) The rice (oryza sativa l.) lesion mimic resembling, which encodes an aaa-type atpase, is implicated in defense response. Mol Genet Genomics 290:611–622CrossRefGoogle Scholar
- Feng ZW, Wang N, Zhu HH, Yao Q (2017) Influences of low pH on the arbuscule development and phosphorus uptake of Rhizophagus intraradices. Mycosystema 36:950–962Google Scholar
- Gao S, Liu T, Li Y, Wu Q, Fu K, Chen J (2012) Understanding resistant germplasm-induced virulence variation through analysis of proteomics and suppression subtractive hybridization in a maize pathogen Curvularia lunata. Proteomics 12:3524–3535. https://doi.org/10.1002/pmic.201200105 CrossRefGoogle Scholar
- Gebauer P, Korn M, Engelsdorf T, Sonnewald U, Koch C, Voll LM. (2017). Sugar Accumulation in Leaves of Arabidopsissweet11/sweet12Double Mutants Enhances Priming of the Salicylic Acid-Mediated Defense Response. Front Plant Sci 8:1378Google Scholar
- Kamoun S (2006) A catalogue of the effector secretome of plant pathogenic oomycetes. Annu Rev Phytopathol 44(1):41–60. https://doi.org/10.1146/annurev.phyto.44.070505.143436 CrossRefGoogle Scholar
- Louis B, Waikhom SD, Roy P, Bhardwaj PK, Singh MW, Goyari S, Sharma CK, Talukdar NC (2014) Secretome weaponries of cochliobolus lunatus interacting with potato leaf at different temperature regimes reveal a cl[xxxx]lhm-motif. BMC Genomics 15:213. https://doi.org/10.1186/s12864-015-1337-3 CrossRefGoogle Scholar
- Pen Y, Chen X, Yang J, Xue M, Wang D, Huang J, Peng Z, Xu J (2011) PacC mediated adaptation to alkaline pH is critical for developing infection hyphae in penetrated plant cells in Magnaporthe oryzae. Phytopathology 101:S140–S140Google Scholar
- Xu S, Chen J, Liu L, Wang X, Huang X, Zhai Y (2007) Proteomics associated with virulence differentiation of curvularia lunata in maize in China. Acta Bot Sin 49:487–496Google Scholar
- Yan J, Liang X, Zhang Y, Li Y, Cao X, Gao J (2017) Cloning of three heat shock protein genes (HSP70, HSP90α and HSP90β) and their expressions in response to thermal stress in loach (Misgurnus anguillicaudatus) fed with different levels of vitamin C. Fish Shellfish Immunol 66:103–111CrossRefGoogle Scholar
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.