Overexpression of OsGF14f Enhances Quantitative Leaf Blast and Bacterial Blight Resistance in Rice

Abstract

Although it is known that rice 14-3-3 family genes are involved in various defense responses, the functions of OsGF14f in response to diseases have not been reported. Here, we showed that the transcription of OsGF14f was significantly induced by leaf blast infection, and the overexpression of OsGF14f quantitatively enhanced resistance to leaf blast and bacterial blight in rice. Further analysis showed that the expression levels of salicylic acid (SA) pathway-associated genes (PAL1, NH1, PR1a and PR10) in the OsGF14f-overexpressing plants, were higher than those in wild-type plants after inoculation with the blast isolate (Magnaporthe oryzae Barr). In addition, the expression level of OsGF14f was significantly induced after SA treatment, and higher endogenous SA levels were observed in the OsGF14f-overexpressing plants compared with that in wild-type plants, especially after blast challenge. Taken together, these results suggest that OsGF14f positively regulates leaf blast and bacterial blight resistance in rice via the SA-dependent signaling pathway.

1. Introduction

As sessile organisms, plants are continuously subjected to various types of stresses such as pathogen attacks, insect herbivory, and environmental stresses [1]. These stresses have adverse effects on plant growth and seed production. To survive, plants have developed intricate mechanisms to efficiently perceive external signals and to tailor their responses to the precise environmental conditions encountered [1,2]. Many genes were reported as important regulators in various responses to stresses [2]. Among these genes, the genes encoding 14-3-3 proteins are modulated by several biotic and abiotic stresses [3,4].

14-3-3 proteins belong to a family of regulatory proteins that are uniquely eukaryotic, and evolutionarily conserved across all eukaryotes [5]. In plants, 14-3-3s have long been thought to play important roles in defense responses [6,7,8,9,10,11]. For example, early research indicated that 14-3-3 genes are differentially modulated during various disease defense responses [6,7,12]. Additionally, 14-3-3s are also involved in R-gene mediated plant disease resistance, as well as in reactive oxygen species (ROS)-mediated plant defense responses [9,13,14,15]. In addition, 14-3-3s have been implicated in programmed cell death (PCD) in tomatoes through directly interacting with mitogen-activated protein (MAP) kinase kinase kinase (MAPKKKa) and SIMKK2 [8,16]. Recently, 14-3-3 proteins have been reported to be the targets of bacterial effectors, and the interactions between 14-3-3 proteins and effectors affect bacterial virulence [10,17,18].

Rice (Oryza sativa L.) is the staple food for over half of the world population, but its production and quality are severely threatened by various diseases. Bacterial blight and blast, caused by the bacteria Xathomonas oryzae pv. oryzae (Xoo) and the fungus Magnaporthe oryzae Barr (M. oryzae), respectively, are the most destructive rice diseases, and can lead to a 30%-50% yield loss when these diseases become epidemic [19]. In our previous studies, we found that the overexpression of OsGF14e enhances resistance to panicle blast, whereas silencing OsGF14e results in increased susceptibility to panicle blast in rice [20]. OsGF14b positively regulates panicle blast resistance but negatively regulates leaf blast. Further results showed that OsGF14b is a target of WRKY71, which positively regulates rice resistance to panicle blast [21]. Moreover, a multi-omics study revealed that OsGF14b strongly activated the gibberellin, auxin and jasmonic acid signaling pathways during pathogen infection [22]. An earlier study demonstrated that OsGF14f was significantly up-regulated during the rice–M. oryzae and rice–Xoo interactions [4], suggesting OsGF14f may play important roles in biotic stresses in rice. However, all these suggested roles are based on its changes in expression level, and the actual functions of OsGF14f in biotic stresses in rice remain to be confirmed.

In the present study, the biological functions of OsGF14f in leaf blast and bacterial blight resistance and the underlying molecular mechanisms were investigated using OsGF14f-overexpressing rice plants and their wild-type plants. Our results suggest that OsGF14f functions as a positive regulator to modulate disease resistance in a quantitative manner, and OsGF14f-mediated disease resistance is associated with the activation of the SA signaling pathway.

2. Results

2.1. Expression Patterns of OsGF14f and Subcellular Localization of OsGF14f

To provide insight into the function of OsGF14f during different physiological processes, tissue-specific expression pattern was examined in various tissues. As shown in Figure 1A, OsGF14f was ubiquitously expressed in all tested tissues, with the highest expression in 14-day-old leaf, and the lowest expression in 10-15 cm panicle. To understand the functions of OsGF14f in blast resistance, quantitative RT–PCR analysis was performed to monitor OsGF14f gene expression at 6, 12, 24 and 48 h after leaf blast inoculation. The qRT–PCR data showed that the transcription of OsGF14f was rapidly and significantly induced by the infection of the blast pathogen (Figure 1B). To examine the subcellular localization of OsGF14f, we transiently co-expressed a GF14f-GFP fusion construct with a nuclear marker (NLS-mCherry) in Arabidopsis protoplasts. As shown in Figure 1C, GF14f-GFP was mainly localized in the cytoplasm, with some occurrence in the nucleus (Figure 1C).

2.2. Overexpression of OsGF14f Enhances Resistance to Leaf Blast in Rice

To confirm the function of OsGF14f in blast resistance in rice, transgenic rice plants constitutively overexpressing OsGF14f (OXGF14f), under the control of the CaMV 35S promoter, were produced in Nipponbare. Three independent homozygous lines (OX-2, OX-3 and OX-5), with increased transcription of GF14f were selected for disease evaluation (Figure 2A). We inoculated 2-week-old OXGF14f plants with the blast isolate GD08-T13, which showed strong virulence to Nipponbare plants, by the spray-inoculation method. The diseased leaf area ranged from 29.61% to 56.02% (average, 39.23%) in transgenic plants, significantly lower than that in wild-type plants (77.35%) (p < 0.01) (Figure 2B,C). To precisely quantify the OsGF14f-mediated quantitative blast resistance, we inoculated the OXGF14f plants with the same isolate by the punch method. Our results showed that the lesions on the leaves of OXGF14f plants were significantly smaller than that on the leaves of Nipponbare plants (p < 0.01) (Figure 2D,E). Moreover, the spore number on the leaves of OXGF14f plants was also significantly less than that on Nipponbare leaves (p < 0.01) (Figure 2F). These results suggest that the overexpression of OsGF14f can improve quantitative resistance against leaf blast in rice.

2.3. Overexpression of OsGF14f Enhances Resistance to Bacterial Blight in Rice

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo), is another devastating rice disease worldwide. Previous research has suggested that blast resistance and bacterial blight resistance might share common pathways to some extent [23]. To see if the OsGF14f gene also functions in bacterial blight resistance, we evaluated the bacterial blight resistance of the OXGF14f plants by inoculation with an isolate from Chinese Xoo race 4. The results showed that the OXGF14f plants showed significantly enhanced resistance to Chinese Xoo race 4 (p < 0.01) (Figure 3A), with the lesion area percentage ranging from 14.9 to 18.3% versus 30% for Nipponbare (Figure 3B). The growth rate of bacteria on the leaves of OXGF14f plants was much slower than that of the wild-type plants at 16 days after inoculation (Figure 3C). These results suggest that the overexpression of OsGF14f enhances quantitative resistance to bacterial blight in rice.

2.4. OsGF14f Positively Regulates the SA-Dependent Pathway Instead of JA Pathway

Jasmonic acid (JA) and salicylic acid (SA) are the major defense signaling compounds mediating disease resistance in plants [24]. To determine whether these signaling compounds are involved in OsGF14f-mediated defense responses, we analyzed the expression patterns of some well-characterized defense-related genes, which are associated with JA- or SA-dependent pathways, in both the Nipponbare and the OsGF14f transgenic plants before and after blast infection. Two LOX (lipoxygenase, LOX1 and LOX11) genes and AOS2 (allene oxide synthase 2), which are involved in the JA-dependent pathway, and two pathogenesis-related PR (PR1a and PR10) genes, NHI (Arabidopsis NPR1 homolog 1) and PAL1 (phenylalanine ammonia-lyase), which are associated with the SA-dependent pathway [25] were selected for the study. The blast pathogen infection strongly induced the expression of PAL1, NH1, PR1a and PR10, both in Nipponbare and in OsGF14f-overexpressing plants (Figure 4). The expression levels of PAL1, PR10 and PR1a were significantly higher in OXGF14f plants than in Nipponbare plants, either before or after infection (p < 0.01) (Figure 4). Although the expression level of NH1 was not significantly changed in the transgenic plants compared to that in control plants before inoculation (Figure 4), its transcript was strongly induced in OXGF14f plants compared to that in control plants after inoculation (Figure 4). There was no obvious difference in the expression levels of LOX1, LOX11 and AOS2 between transgenic plants and wild-type plants, either before or after pathogen inoculation (Supplemental Figure S1). These results suggest that OsGF14f-mediated blast resistance may be involved in SA-dependent instead of JA-dependent signaling pathways.

2.5. OsGF14f Is Induced by SA Treatment and Overexpression of OsGF14f Can Increase Endogenous SA Levels

To further support the involvement of OsGF14f in SA-dependent signaling pathways, exogenous SA treatment was applied, and the expression changes of OsGF14f in wild-type plants at different time points (3, 6, 12 and 24 h) were monitored using qRT-PCR. Our results showed that the transcription levels of OsGF14f increased at all four time points after SA treatment in Nipponbare, with the peak at 24 h (Figure 5). In addition, we also observed that the endogenous SA level in OXGF14f plants was significantly increased after blast infection, while it remained at the same level in wild-type plants; and the SA level was significantly higher (p < 0.05) in OXGF14f plants than that in wild-type plants, both before and after blast infection (Figure 6). Together, these results suggest that OsGF14f positively regulates the SA-dependent pathway.

3. Discussion

3.1. OsGF14f Enhances Blast and Bacterial Blight Resistance in Rice in a Quantitative Manner

It is well-known that plants fight against blast invasion via two different resistance strategies: qualitative (complete) resistance, mediated by major disease resistance (R) genes, and quantitative (partial) resistance, contributed by multiple genes or quantitative trait loci (QTL) [26,27]. In most cases, qualitative resistance mediated by R genes is highly efficient, but it is race-specific and easily overcome, owing to the rapid evolution of pathogens [27,28]. In contrast, quantitative resistance conferred by quantitative trait loci (QTL) is presumably non-race-specific and is generally considered to be more broad-spectrum and durable under natural conditions [26]. Thus, quantitative resistance has been considered as a preferred strategy in disease control in rice [29]. Over the past two decades, numerous QTLs for quantitative blast and bacterial blight resistance have been identified. However, these sources have not been effectively used for the improvement of blast and bacterial blight resistance in rice because the genes of underlying resistance QTLs are unknown. Therefore, the identification and isolation of the genes of underlying resistance QTLs is the key for effective molecular breeding for quantitative disease resistance. In the present study, our results showed that enhanced disease resistance correlated with increasing transcription levels of OsGF14f (Figure 1, Figure 2 and Figure 3). Moreover, the OsGF14f-overexpressing rice plants displayed less diseased leaf area, smaller lesion size, fewer spores during blast infection, lower percent lesion length and fewer spores during bacterial blight infection compared with the wild-type plants (Figure 2 and Figure 3), exhibiting the nature of quantitative disease resistance. These results suggest that OsGF14f confers blast and bacterial blight resistance in a quantitative manner, and is a good target in molecular breeding for durable disease resistance in rice.

3.2. OsGF14f-Mediated Disease Resistance Is Involved in the SA-Dependent Pathway

SA is an archetypal defense hormone and its importance in the hard wiring of the plant innate immune system is well documented, particularly in the model plant Arabidopsis [30]. In this study, we showed that the expression levels of the defense-related genes, including PAL1, NH1, PR1a and PR10 which are involved in the SA-dependent pathway, were significantly higher in OsGF14f transgenic plants than in Nipponbare plants, both before and after pathogen infection (Figure 4). Moreover, the transcript of OsGF14f was significantly induced by exogenous SA, and the endogenous SA levels were significantly higher (p < 0.05) in OXGF14f plants than those in wild-type Nipponbare plants, both before and after fungal infection (Figure 5 and Figure 6). These results suggest that OsGF14f mediated disease resistance is involved in the SA-dependent pathway. This is consistent with the previous findings, that the resistance to biotrophic and hemi-biotrophic pathogens is frequently controlled by the SA-dependent pathway [31]. We also found that the expression of LOX1, LOX11 and AOS2 is not changed between transgenic plants and wild-type plants, either before or after pathogen inoculation (Supplemental Figure S1), indicating that the JA-dependent pathway might function scarcely in OsGF14f-mediated disease resistance. Interestingly, in our previous study, we identified that OsGF14b, another member of the 14-3-3 family, was induced by blast infection and positively regulates panicle blast resistance in rice [20]. However, OsGF14b-mediated disease resistance is associated with the activation of the JA-dependent pathway and suppression of the SA-dependent pathway [21], different to the OsGF14f-mediated disease resistance pathway. These results suggest that there might be divergent functions and regulatory mechanisms in disease resistance among the members of the 14-3-3 family.

In conclusion, we have confirmed the functions of OsGF14f in response to blast and bacterial blight infection through gene expression analysis, a transgenic method and physiology analysis, and its possible regulatory mechanisms underlying these processes were also investigated. Our results indicate that OsGF14f positively regulates leaf blast and bacterial blight resistance in rice. OsGF14f-mediated disease resistance is dependent on the SA signaling pathway. Since blast and bacterial blight are two of the most devastating diseases in rice, and quantitative disease resistance is more durable, OsGF14f is a good target in rice improvement for disease resistance.

4. Materials and Methods

4.1. Plant Materials and Pathogens

The OsGF14f-overexpressing rice plants and their wild-type plants, Nipponbare, were used in this study. Rice seeds were surface-sterilized and transferred to Murashige and Skoog medium [32] and incubated in a growth chamber under light of 200 μmol/m²/s with a 12 h photoperiod at 26 °C. After germination, rice seedlings were transplanted into soil and kept in a greenhouse. Blast (M. oryzae) isolate GD08-T13 collected from Guangdong, China, was used for rice blast inoculation, and Chinese Xoo race 4 isolate collected from Guangdong, China, was used for bacterial blight inoculation.

4.2. Pathogen Inoculation and Evaluation of Disease Resistance

The inoculum of M. oryzae isolate GD08-T13 was prepared as described by Liu et al. (2018) [33]. The revived isolate GD08-T13 was cultured on prune agar plates (three pieces prune, 5 g lactose, 1 g yeast extract, and 20 g agar bar in 1 L, pH of 6–6.5, autoclaved) for 7 d at room temperature, and mycelial growth was scraped with a sterilized spatula and exposed to fluorescent light for 4–5 days to induce sporulation. Spores were collected and suspended in water to reach a concentration of 1 × 10⁶ mL⁻¹. To examine the effect of rice blast inoculation on the expression of OsGF14f, conidia of GD08-T13 were washed and suspended at 1 × 10⁶ cells mL⁻¹ in sterile water, and sprayed on the 14-day-old Nipponbare plants, which were then incubated at 26 °C in a growth chamber (16,000 Lux and 100% relative humidity) in the dark for 24 h, followed by 12 h light and 12 h dark cycles.

Two inoculation methods were used in evaluation of blast resistance in this study. For spray inoculation, two-week-old rice seedlings were sprayed with a spore suspension of GD08-T13 (1 × 10⁶ spores/mL) containing 0.05% Tween-20. Inoculated plants were maintained in the same growth chamber used for transcription analysis at 26 °C, 16,000 Lux and 100% relative humidity in the dark for 24 h. Subsequently, the growth chamber was set to a photoperiod of 2 h light and 12 h dark at 26 °C and 100% relative humidity. Disease was assessed 6 d after inoculation by measuring the diseased leaf area. For punch inoculation, six-week-old plants were punched as described previously [34,35]. Briefly, 5 μL of spore suspension of GD08-T13 (5 × 10⁵ spores/mL) containing 0.05% Tween-20 was added to the press-injured spots on fully expanded rice leaves; the inoculated spots were wrapped with transparent Scotch tape, and inoculated leaves were photographed at 14 days after inoculation, and lesion size was measured using ImageJ (http://rsbweb.nih.gov/ij/, accessed on 8 July 2021). For determination of in planta sporulation after punch inoculation, leaf strips containing a lesion spot were excised and submerged in 100 μL of distilled water, in a 1.5 mL microcentrifuge tube. After the suspension was vigorously mixed, spores were counted with a microscope. To evaluate bacterial blight disease, plants were inoculated with Chinese Xoo race 4 (OD₆₀₀ = 0.3) isolate at the booting stage by the leaf-clipping method [36]. Disease was scored by measuring the percent lesion length (lesion length/leaf length) at 12 d after inoculation.

4.3. Plasmid Construction and Rice Transformation

To generate the overexpression vector of OsGF14f, we obtained the full-length coding region of the gene from Nipponbare leaf cDNA by PCR using primers GF14f-OE-F/R (Supplemental Table S1), and cloned the fragment into the PHQSN (modified from pCAMBIA1390), harboring a CaMV 35S promoter. The plasmid was transferred into the calli induced from the immature seeds of Nipponbare, using the Agrobacterium tumefaciens-mediated rice transformation method as previously described [37].

4.4. Subcellular Localization

The full-length cDNA of OsGF14f was constructed into the pCAMBIA1300-GFP to generate pUBQ10: OsGF14f-GFP construct. The OsGF14f-GFP and NLS-mCherry plasmid were co-transformed into Arabidopsis protoplasts for transient expression following previously described method [38]. After incubation in darkness for 16 h, images were captured using a confocal fluorescence microscope (LSM 710).

4.5. Gene Expression Analysis

Total RNA was extracted from rice tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and was treated with DNaseI (Takara, Dalian, China) to remove genomic DNA contamination. The first strand of cDNA was synthesized from 1 μg of total RNA using the primescript^TM RT reagent kit (Takara, Dalian, China) according to the manufacturer’s instructions. Quantitative PCR was performed using SYBR Premix ExTaq^TM (Takara, Dalian, China) on a CFX Connect real-time PCR detection system (Bio-Rad, Hercules, CA, USA), and data were analyzed using Bio-Rad CFX Manager 3.1 (Bio-Rad, Hercules, CA, USA). EF1a was used as the internal control. Gene-specific primers that were used are listed in Supplemental Table S1.

4.6. SA Treatment and Measurement

To examine the effect of rice blast inoculation on the expression of OsGF14f, two-week-old rice seedlings were sprinkled with 100 μM hormone solution, and sampled at 3 h, 6 h, 12 h and 24 h after treatment.

The hormones SA were extracted from the rice leaf [39], and then 10 μL of SA sample solution was injected onto a C18 column (AQUITY UPLC BEH 130, 1.7 μm, 2.1 by 100 mm, Waters) at a flow rate of 0.1 mL/min, and the column was maintained at 30 °C. The sample solution was separated by reversed-phase ultra-fast LC (Shimadzu, Kyoto, Japan) with a multi-step linear gradient elution over 30 min. The eluate was then introduced into the electrospray ion source of a tandem triple quadrupole MS analyzer (API4000, AB SCIEX, Foster City, CA, USA), and the SA compound was quantified in the multiple reaction monitoring (MRM) mode using optimized MS/MS conditions, which are listed in Supplemental Table S2. The Analyst 1.5.2 software (AB SCIEX, Foster City, CA, USA) was used to control the instrument, and to acquire and process all of the MS data.

4.7. Statistical Analysis

All the graphic data are presented as mean  ±  SD. Student’s t-test was used to test the significance of difference: * and ** indicate a significant difference at p  <  0.05 and p  <  0.01, respectively.