Research | Open | Published:
A core effector UV_1261 promotes Ustilaginoidea virens infection via spatiotemporally suppressing plant defense
Phytopathology Researchvolume 1, Article number: 11 (2019)
False smut is a destructive grain disease of rice worldwide, characterized by false smut balls formed in rice flowers. Here we identified a small secreted protein UV_1261 contributing to virulence of Ustilaginoidea virens, the causal agent of this disease. The sequence of UV_1261 was highly conserved among isolates of U. virens and absent in other fungi. UV_1261 encodes a protein targeted to plant chloroplasts. Its expression exhibited a bimodal pattern during pathogenesis. Ectopic expression of UV_1261 in Nicotiana benthamiana and Arabidopsis led to suppression of flg22-induced ROS burst, callose deposition, and expression of defense-related genes, as well as enhanced susceptibility to powdery mildew in Arabidopsis. Down-regulation of UV_1261 via exogenous siRNA treatment resulted in reduced number of false smut balls. Consistently, stably knocking-down UV_1261 caused less number of false smut balls associated with higher expression of defense-related genes in rice flower. Taken together, our data demonstrate that UV_1261 is a core effector of U. virens essential for virulence and suppressing defense in rice flower, and thus may serve as a potential molecular target for controlling rice false smut disease.
Flower is a nutrient-rich sink organ, attracting habitation of a large number of microorganisms. Flower-infecting fungi have caused diseases in many economically important crops. For example, Claviceps purpurea causes ergot disease in rye and Gibberella zeae causes Fusarium head blight in wheat (Ngugi and Scherm 2006). In recent years, rice false smut (RFS) disease, caused by the flower-infecting pathogen Ustilaginoidea virens, has emerged as a serious grain disease worldwide (Fan et al. 2016). Occurrence of RFS disease not only leads to yield loss, but also contaminates grains and straws with mycotoxins that are poisonous to both human and animals (Koiso et al. 1994; Nakamura et al. 1994). However, our understanding on the pathogenesis of this disease is very limited.
In recent years, several groups independently reported the infection process of U. virens in rice flower and a few genes have been identified to be involved in the process (Ashizawa et al. 2012; Tang et al. 2013; Hu et al. 2014; Fan et al. 2015; Song et al. 2016). Generally, the infection process can be divided into two stages. In stage I, conidia of U. virens germinate on the surface of rice spikelet, and generate hyphae or mycelia that grow epiphytically (Ashizawa et al. 2012; Fan et al. 2012). In stage II, mycelia extend into the inner space of spikelet via the gap between the lemma and the palea (Ashizawa et al. 2012), firstly attack stamen filament (Tang et al. 2013), then infect lodicule, stigma and style (Song et al. 2016; Yong et al. 2016), and ultimately embrace all the floral organs to form a ball-shape colony called false smut ball (Tang et al. 2013; Fan et al. 2015). Stage I lasts for 3–5 days, while stage II 7–10 days (Tang et al. 2013; Fan et al. 2015). In addition, a few genes have been found to be associated with infection of U. virens in rice. For example, UvPRO1 encodes a C6 transcription factor, and disruption of the gene resulted in reduced growth rate, inability to sporulate, increased sensitivity to abiotic stresses, and abolishment of virulence (Lv et al. 2016). UvSUN2 encodes a SUN domain-containing protein, and mutation in UvSUN2 led to altered cell wall construction, abnormal fungal growth and stress responses, as well as inability to infect rice (Yu et al. 2015). On the contrary, knock-out or knock-down of Uvt3277, a gene encoding a putative low-affinity iron transporter, enhanced U. virens virulence (Zheng et al. 2017). Moreover, decoding the U. virens genome opens the door for investigation of effectors involved in U. virens pathogenesis (Zhang et al. 2014).
Effectors of a pathogenic microbe have versatile roles in manipulating host immunity to promote infection of the pathogen. They act either in interfering with phytohormone and secretory pathways, suppressing the pattern recognition receptor-mediated surveillance system, targeting plasma membrane components and chloroplasts, or generating a microenvironment favorable to infection (Deslandes and Rivas 2012; Dou and Zhou 2012; Kazan and Lyons 2014; Presti et al. 2015). For example, Phytophthora sojae effector PsAvr3c can reprogram soybean pre-mRNA splicing system to defeat host immunity and promote infection (Huang et al. 2017). Pseudomonas syringae pv. tomato injects effectors HopM1 and AvrE1 into Arabidopsis leaves to establish an aqueous space for successful infection (Xin et al. 2016). P. sojae secretes a paralogous decoy PsXLP1 to bind host GmGIP1, thus releasing the apoplastic effector PsXEG1 to promote infection (Ma et al. 2017). U. virens genome encodes at least 193 putative effector proteins (Zhang et al. 2014). Thirteen of them can induce cell death and 18 can suppress plant hypersensitive responses in Nicotiana benthamiana or rice protoplast (Zhang et al. 2014; Fang et al. 2016). However, up to date, none of them has been in-depth investigated during U. virens infection.
In an earlier de novo transcriptome analysis (Fan et al. 2015), we detected a full-length candidate effector gene Uv2169 whose transcription was significantly up-regulated upon U. virens infection. Uv2169 was coincidently reported as UV_1261 that can suppress Burkholderia glumae-induced cell death in N. benthamiana (Zhang et al. 2014). Thereafter, we renamed Uv2169 as UV_1261. Here, we examined its expression pattern during infection and tested the effects of knock-down or overexpression of UV_1261. The results demonstrated that UV_1261 specifically suppressed plant defense responses in rice spikelets to facilitate colonization of U. virens.
The expression pattern of UV_1261 over U. virens infection of rice panicles
To understand how UV_1261 facilitates U. virens infection, we examined its expression pattern during U. virens infection by a time-course analysis. To this end, we exploited a compatible U. virens-rice interaction established in our previous studies with the isolate PJ52 and the rice accession Pujiang 6 (Fan et al. 2015; Huang et al. 2016). Sequence analysis showed that UV_1261 in PJ52 was identical to that in the published UV-8b genome (Additional file 1: Figure S1) (Zhang et al. 2014). Compared to its expression in PSB medium, UV_1261 was up-regulated at 1–3 days post inoculation (dpi), and then slightly decreased from 5 to 9 dpi. It was up-regulated again at 11 dpi, and reached to the highest expression at 13 dpi, forming a bimodal pattern (Fig. 1). This bimodal pattern is coincident with the two infection stages of U. virens (Fan et al. 2016), during which no obvious symptoms were detected from 1 to 7 dpi; whereas white fungal mass were seen in inner space of a spikelet at 9 dpi and the fungal mass increased to protrude out of the spikelet at 15 dpi (Fan et al. 2015). Therefore, the expression pattern of UV_1261 suggested its role in both infection stages.
To check whether UV_1261 expression was associated with rice defense response, we examined the expression patterns of some defense-related genes, including OsNAC4, OsPR1#012, OsPR10b, and OsBETV1 (Li et al. 2014b; Fan et al. 2015) over the infection of PJ52 by RT-qPCR. Intriguingly, OsNAC4 showed an expression pattern reverse to that of UV_1261 (Fig. 1). Particularly, OsNAC4 was in low expression at 1, 3, 11 and 13 dpi, when UV_1261 was highly expressed; whereas, OsNAC4 was greatly induced from 5 to 9 dpi, when UV_1261 was at lower expression. OsPR1#012 and OsPR10b were induced at early time points, but down-regulated at later time points, especially at 13 dpi when UV_1261 reached the highest expression (Fig. 1). By contrast, OsBETV1 was up-regulated across the infection process, although to a lesser extent at later time points (Fig. 1). These results imply that rice defense response is induced at earlier time points but then suppressed by infection of U. virens.
UV_1261 encodes a small secreted cysteine-rich protein
UV_1261 contains a 393 bp codon, encoding a small cysteine-rich protein with 130 amino acid (aa) residues (Fig. 2a). The first 23 aa residues was a predicted signal peptide. First, we verified the function of the predicted signal peptide via an invertase secretion assay following previous studies (Klein et al. 1996; Jacobs et al. 1997; Oh et al. 2009; Cheng et al. 2017). To this end, we cloned the sequence encoding the first 23 aa residues of UV_1261 to the N-terminus of the mature yeast invertase gene SUC2 and transformed into YTK12, a SUC2-deficient yeast strain. Similarly, we made constructs with the sequences encoding the first 25 aa residues of Magnaporthe oryzae Mg87 and the validated signal peptide of P. sojae effector Avr1b as negative and positive control, respectively (Gu et al. 2011; Cheng et al. 2017). Then, the constructs were transformed into YTK12 for invertase secretion assay. As expected, all the three constructs could enable growth of YTK12 on CMD-W medium where yeast can grow without invertase secretion. However, only the constructs expressing SUC2 fused with the signal peptide of UV_1261 and Avr1b could enable YTK12 growth on YPRAA medium where yeast grow requires signal peptide-mediated secretion of the invertase (Fig. 2b). The negative control peptide of Mg87 could not enable YTK12 growth on YPRAA medium. These data indicate that the predicted signal peptide of UV_1261 is functional in mediating secretion.
UV_1261 is mainly localized in the cytoplasm and the chloroplast
To determine the subcellular localization of UV_1261 in planta, we cloned the sequence encoding mature UV_1261 (without signal peptide) at the N-terminus of eYFP for transient expression in N. benthamiana and stable expression in transgenic Arabidopsis Col-gl. Transient expression showed that UV_1261-eYFP was localized in the cytoplasm, the nucleus and the chloroplasts of N. benthamiana cells (Additional file 2: Figure S2a-c). In transgenic Arabidopsis plants, UV_1261-eYFP was mainly aggregated as dots, in addition to scattered distribution in the cytoplasm (Fig. 2c). Intriguingly, the fluorescent signal of UV_1261-eYFP was overlapped with the auto-fluorescent signal of the chloroplasts, indicating localization of UV_1261 in the chloroplasts (Fig. 2d). Moreover, UV_1261-eYFP fusion protein was intact as confirmed by Western blot (Fig. 2e). Taken together, these data indicate that UV_1261-eYFP is localized in the cytoplasm and the chloroplasts.
UV_1261 suppresses basal defense responses in N. benthamiana and Arabidopsis
PAMPs like flg22 can trigger basal defense responses, such as rapid burst of ROS, callose deposition and expression of defense-related genes. Thus, we tested whether UV_1261 played a role in suppression of flg22-triggered defense responses. When UV_1261 was transiently expressed in N. benthamiana, flg22-triggered ROS burst was obviously inhibited (Additional file 2: Figure S2d). When UV_1261 was stably expressed in Arabidopsis, flg22-induced ROS accumulation was also inhibited (Fig. 3a). Meanwhile, flg22-induced callose deposition was remarkably reduced in UV_1261-eYFP transgenic line (Fig. 3b), being about one-third of that in the wild-type Col-gl. Next, RT-qPCR was performed to examine the expression of defense-related genes in Arabidopsis (Li et al. 2018), including FLG22-INDUCED RECEPTORLIKE KINASE 1 (FRK1, At2g19190), WRKY29 (At2g23550) and the PATHOGENESIS-RELATED1 (PR1, At2g19990). The expression of FRK1 and WRKY29 was highly induced as early as 3 hours post application (hpa) of flg22 in Col-gl, while PR1 was induced and reached to the highest expression at 12 hpa (Fig. 3c). By contrast, the induction of all the three tested genes were much lower in UV_1261-eYFP transgenic line than in Col-gl (Fig. 3c). Taken together, UV_1261 could suppress basal defense responses in N. benthamiana and Arabidopsis.
UV_1261 increases infection of tobacco powdery mildew in Arabidopsis
Modulation of basal defense responses by UV_1261 prompted us to test whether it can compromise disease resistance against biotrophic pathogen. To this end, we inoculated the tobacco powdery mildew strain Golovinomyces cichoracearum SICAU1 onto six-week-old plants of UV_1261-eYFP transgenic line and Col-gl. The results showed that UV_1261-eYFP leaves sustained more white fungal mass and spores than Col-gl leaves at 12 dpi (Fig. 3d, e). Trypan blue staining displayed that there were more hyphae in UV_1261-eYFP than in Col-gl leaves (Fig. 3f). These data indicate that UV_1261 promotes the powdery mildew infection in Arabidopsis.
Exogenous double-stranded siRNA of UV_1261 reduces U. virens virulence
Since exogenous application of double-stranded RNAs can trigger gene silencing (Fire et al. 1998), we employed this approach to preliminarily test whether UV_1261 was involved in U. virens virulence. To this end, we synthesized a 21 bp double-stranded siRNA specifically targeting UV_1261 and prepared inocula using the strain P4 cultured on media containing 10 nM/mL of the synthesized siRNA. As expected, in the inocula from the siRNA-containing media, the expression of UV_1261 was reduced to as low as 50% of that in control (Fig. 4a), indicating that the siRNA is functional in silencing of UV_1261. Then the inocula were injected into more than 30 rice panicles at late booting stage in comparison with control inocula. After 28 dpi, the number of false smut balls was counted (Fig. 4b). Typically, the number of false smut balls per inoculated panicle varied from less than 10 to more than 50 (Fig. 4b). This is a common phenomenon to rice false smut disease that makes disease assay difficult. To evaluate the effects of different inocula, we classified the diseased panicles into five types according to the number of RFS balls per panicle, i.e. 1–10, 11–20, 21–40, 41–50 and > 50. The data demonstrated that nearly half (47%) of the diseased panicles showed mild infection with the number less than 10 in siRNA-treated group, but less than one third (31%) in control group (Fig. 4c). On the contrary, the percentage of severely diseased panicles (> 20 balls per panicle) was much lower in siRNA-treated group than in control (Fig. 4c). These data indicate that suppression of UV_1261 in vitro could reduce U. virens virulence.
Silencing of UV_1261 attenuates U. virens virulence
To further confirm the role of UV_1261 in U. virens virulence, we tried to knockout UV_1261 by using gene replacement strategy, but failed to obtain any knockout mutants probably due to low homologous recombination frequency in U. virens (Zheng et al. 2016). We then tried to silence UV_1261 in U. virens isolate PJ52. Fortunately, we obtained positive transformants with reduction of UV_1261 expression. Subsequently, three independent transformants (T1, T2 and T3) with significantly lower expression of UV_1261 were selected for further analysis (Fig. 5a). All the three transformants formed colonies with diameters similar to that of the wild-type PJ52 after cultured for 2 weeks on PSA, and produced conidia with unaltered morphology (Fig. 5b). To test their virulence, the transformants were inoculated into rice panicles of cultivar Pujiang 6 that is highly susceptible to U. virens (Huang et al. 2016). Then, the number of RFS balls in each diseased panicle was recorded at 4 weeks post inoculation (wpi). The results showed that PJ52 and the three transformants all successfully infected rice panicles, with the rate of diseased panicle reaching 100%. PJ52 formed more than 110 RFS balls in average in the inoculated panicles (Fig. 5c). However, the three transformants formed much less number of RFS balls than PJ52 (Fig. 5c, d). In addition, we observed that the expression of UV_1261 positively correlated with the virulence of U. virens (Fig. 5). Therefore, these data confirmed that UV_1261 is a virulence effector of U. virens.
Silencing of UV_1261 leads to higher expression of rice defense-related genes in U. virens-inoculated spikelets
To test whether knockdown of UV_1261 influenced the induction of rice defense, we inoculated UV_1261-silenced transformants (T1 and T3) into rice panicles at booting stage with PJ52 as control, and examined the expressions of four rice defense-related genes at 5 and 9 dpi. Our data showed that the expression of OsNAC4 remained unchanged at 5 dpi, but was significantly higher in T1 and T3-inoculated spikelets than in PJ52-inoculated control at 9 dpi (Fig. 6). The expression of OsPR10b at 5 dpi was increased to about 9-fold in T1 or T3-inoculated spikelets compared with that in control, and nearly 4-fold at 9 dpi (Fig. 6). Similar trend was shown for OsPR1#012. The expression of OsBETV1 was increased to 2–4 fold in T1 or T3-inoculated spikelets at both 5 and 9 dpi. Overall, compared to PJ52-inoculated spikelets, T1 and T3-inoculated samples exhibited significantly higher expression of all four tested genes at either 5 and/or 9 dpi, indicating that UV_1261-silenced strains cannot efficiently suppress the expression of rice defense-related genes. Therefore, these data implied that UV_1261 promotes U. virens infection via suppression of host defense.
UV_1261 has no intra-species diversity
BLAST analysis showed that UV_1261 had no homologs in U. virens genome and no orthologs in any other published fungal genomes, indicating that UV_1261 is a single gene specific in U. virens. Then, evolutionary analysis was performed on 50 U. virens isolates collected from different rice production areas in China (Additional file 3: Table S1). The results showed that these U. virens isolates were classified into five subgroups, and isolates from different locations could be grouped together (Additional file 4: Figure S3a). These isolates were used to amplify the full-length of UV_1261. Sequence analysis revealed that UV_1261 sequences from all the tested isolates were identical (Additional file 4: Figure S3b), suggesting that UV_1261 is extremely conserved among U. virens isolates.
There are 193 putative effectors in U. virens genome (Zhang et al. 2014), none of them was reported to be involved in U. virens pathogenicity. In this study, we demonstrated that UV_1261 is a U. virens-specific effector involved in suppressing plant defense responses and required for full virulence of U. virens.
Fungal effectors are featured by low sequence similarity and lack of conserved motif within and across species (Sperschneider et al. 2015), although some exceptions exist, such as the LysM domain-containing effectors Ecp6 from Cladosporium fulvum and Slp1 from M. oryzae (de Jonge et al. 2010; Mentlak et al. 2012). In addition, fungal effectors are also defined to be secreted from the pathogen and their expression should be induced in planta during pathogenesis (Guyon et al. 2014; Sperschneider et al. 2014). In consistent with these criteria for defining an effector, UV_1261 is a U. virens-specific effector. First, no homologous sequence of UV_1261 was found in U. virens or other fungal genomes. Second, Interpro search revealed no conserved domain in UV_1261 protein. Third, UV_1261 possessed a functional signal peptide at its N-terminus (Fig. 2b), and expression of UV_1261 was significantly induced in U. virens upon infection of rice (Fig. 1).
Knock-out or knock-down a single effector usually does not affect virulence, due to functional redundancy of the effector pool in a pathogen genome (Birch et al. 2008). For instance, silencing of PSTha5a23 in Puccinia striiformis f. sp. tritici did not change its virulence in wheat (Cheng et al. 2017). Knock-out AvrPi9 or other 77 putative effector genes in M. oryzae also did not affect its virulence in rice (Saitoh et al. 2012; Wu et al. 2015). However, deletion of core effectors may compromise the virulence of pathogens. For example, disruption of the core effector pep1 in Ustilago maydis caused inability of the pathogen to infect maize leaf, although the ∆pep1 mutants had normal saprophytic growth (Doehlemann et al. 2009). In this study, silencing of UV_1261 in vitro and in vivo consistently showed reduction of U. virens virulence, indicating that UV_1261 is a core effector (Fig. 4 and Fig. 5).
In addition to photosynthesis and primary metabolism, chloroplast is involved in production of pro-defense molecules, such as SA, JA, ABA, ROS and calcium (Serrano et al. 2016). Pathogens have evolved effectors to interfere with chloroplast function, so as to fight against host immunity. For instance, P. syringae pv. tomato secretes a effector protein HopN1 to interact with PsbQ in thylakoids, resulting in reduction of chloroplastic ROS (Rodriguez-Herva et al. 2012). Pst effectors HopK1 and AvrRps4 also target to host chloroplasts to suppress ROS production, although their targeted proteins are unknown (Li et al. 2014a). Effector proteins Cmu1 from U. maydis (Djamei et al. 2011), PsIsc1 from P. sojae and VdIsc1 from Verticillium dahliae (Liu et al. 2014) modulate SA biosynthesis in plastid and suppress plant immune responses. In the present work, UV_1261 was targeted to chloroplasts (Fig. 2c-e), suggesting its role in interfering with plant immunity, which was further supported by that UV_1261 could suppress basal defenses in N. benthamiana, Arabidopsis and rice (Fig. 3, Fig. 6 and Additional file 2: Figure S2d).
Pathogen effectors are subjected to rapid evolution, so as to evade the recognition by host resistance proteins. For example, insertion of transposable element and somatic exchange in wheat stem rust effector protein AvrSr35 and AvrSr50, respectively, drove the pathogen escaping recognition by R proteins Sr35 and Sr50 (Chen et al. 2017; Salcedo et al. 2017). Mg-SINE insertion in M. oryzae AvrPi9 converted an avirulent isolate to a virulent isolate (Wu et al. 2015). However, in our work, no polymorphism was detected in UV_1261 from around 50 U. virens isolates collected across multiple rice production areas in China (Additional file 4: Figure S3), implying that UV_1261 is of extremely low intraspecific variation.
Overall, UV_1261 is a novel core effector protein that plays an important role in U. virens virulence by suppressing plant defense responses. As UV_1261 is unique and highly conserved in U. virens, it could be a potential molecular target for developing efficient strategies to control RFS disease.
Plant materials and pathogen isolates
Plants of rice cultivar Pujiang 6 were grown in an experimental field under natural conditions. N. benthamiana and Arabidopsis accession Col-gl were planted in a growth room at 10 h light/14 h darkness, 23 °C and 70% relative humidity until subsequent experiments. U. virens isolates were obtained via amerosporous purification from RFS balls. U. virens isolate PJ52 was used for gene expression analysis and U. virens transformation experiments. The GFP-tagged U. virens strain P4 was used for siRNA treating experiment. The powdery mildew Golovinomyces cichoracearum SICAU1 (Zhang et al. 2015) was maintained on leaves of tobacco in a growth room at 16 h light/8 h darkness, 23 °C and 75% relative humidity.
U. virens inoculation
Artificial inoculation of U. virens was performed as described previously (Fan et al. 2015). In brief, U. virens mycelia were cultured in potato-sucrose broth (PSB) at 28 °C and 120 r/min for 5–7 days, and the mixture of mycelia and conidia were blended as inocula, with conidia concentration adjusted to 1 × 106 conidia/mL. At late booting stage of rice (5–7 days before heading), U. virens inocula were injected into rice panicles by a syringe with needle. Mock-inoculation was carried out with PSB. Rice spikelets were collected at 1, 3, 5, 7, 9, 11, 13 and 15 dpi for subsequent experiments. The number of false smut balls was recorded at about 4 wpi.
Total RNA was extracted using TRIzol reagent (Invitrogen) and was reverse transcribed using SuperScriptfirst-strand synthesis kit (Invitrogen). Quantitative RT-PCR (RT-qPCR) was performed using SYBR Green mix (TaKaRa) and gene specific primers (Additional file 5: Table S2). The reference gene UvTub2α was used for expression analysis of UV_1261, OsUbi for rice genes, and AtACT2 for Arabidopsis genes. Comparative CT method 2–ΔΔCT (Livak and Schmittgen 2001) was applied for calculating relative expression.
For validation of UV_1261 signal peptide, the corresponding DNA fragment was amplified with primer pair SP1261_EcoRIF/SP1261_XhoIR, and inserted into pSUC2T7M13ORI (pSUC2) vector at restriction enzyme sites EcoRI and XhoI. For ectopic expression of UV_1261 in N. benthamiana and Arabidopsis, primer pair Uvm1261_KpnIF/Uv1261_KpnIR was used to amplify the full-length of UV_1261 minus signal peptide sequence, and inserted into pCAMBIA1300-eYFP vector with KpnI. For U. virens transformation, 1261_EcoRIF/1261_SpeIR were used to amplify the antisense strand of UV_1261, and inserted into Pzp-bar-Ex vector (Additional file 6: Figure S4) to silence UV_1261.
Yeast invertase secretion assay
pSUC2-derived plasmids were transformed into yeast strain YTK12 with the lithium acetate method (Gietz et al. 1995). The subsequent procedures of yeast secretion assay was performed as described (Fang et al. 2016).
Agrobacterium tumefaciens-mediated transformation
A. tumefaciens strain GV3101 containing UV_1261-eYFP construct was transiently expressed in N. benthamiana as described (Huang et al. 2014) and stably expressed in Arabidopsis Col-gl with floral dipping method (Clough and Bent 1998). A. tumefaciens strain AGL1 containing UV_1261-related constructs were transformed into conidia of PJ52 according to the published protocol with modifications (Yu et al. 2015). In brief, PJ52 conidia were produced in PSB medium at 28 °C, 120 r/min for 7 days. AGL1 containing indicated constucts was cultured in minimal medium at 28 °C, 200 r/min for 48 h, supplemented with 50 μg/mL kanamycin. Then diluted AGL1 (OD600 = 0.15) was pre-cultured in induction medium, supplemented with 50 μg/mL kanamycin and 50 μM acetosyringone, for about 6 h until OD600 reaching 0.25. One hundred microliter of AGL1 cells were mixed with equal volume of 106 conidia/mL PJ52, and incubated on nitrocellulose membrane (Whatman, pore size = 0.45 μm) in co-cultivation medium at 25–28 °C for 72 h. The membrane was then transferred to the selection medium, i.e. potato-sucrose-agar (PSA) medium with 0.003% Basta (Yobios BioTect) and 500 μg/mL Timentin (Solarbio), and incubated at 25–28 °C until transformants appeared. The colonies were again transferred to selection medium to screen for positive transformants, which were further confirmed by PCR with primer pairs UvmitoF/UvmitoR, BastaF/BastaR, and 1261_EcoRIF/TtrpC_R. UV_1261 expression were examined by RT-qPCR with the primer pair UV_1261_F2 /UV_1261_R2. Primer sequences are included in Additional file 5: Table S2.
The construct UV_1261-eYFP or empty vector expressing only eYFP was co-expressed with 2 × RFP-NLS (Huang et al. 2014) in N. benthamiana. Leaves were sampled at 3 days post infiltration for microscopy observation. Fully expanded leaves from T2 generation of transgenic Arabidopsis (5–6 week old) expressing UV_1261-eYFP were sampled and stained in Propidium Iodide (PI) for 10 min before observation. Leaf samples were checked under a confocal laser scanning microscope (Nikon A1). The image data were processed with NIS-Elements viewer and Adobe Photoshop. Western blot was performed with anti-GFP sera (BBI Life Science).
ROS, callose assays and trypan blue staining
Leaves of Col-gl and UV_1261-eYFP-expressing plants were prepared and treated with either ddH2O or 1 μM flg22, and subjected to ROS and callose assays as previously described (Li et al. 2018). Spores of powdery mildew SICAU1 were inoculated onto leaves of Col-gl and transgenic line expressing UV_1261-eYFP. Spore production was determined and pathogen hyphae were stained with trypan blue according to the reported method (Zhao et al. 2015). Images were acquired under a Canon EOS Rebel T2i.
Double-stranded siRNA specifically targeting UV_1261 was designed and synthesized by Shanghai GenePharma Co., Ltd. The sequences are listed in Additional file 5: Table S2. SiRNA was dissolved in DEPC-treated ddH2O and added in PSB to a final concentration of 10 nM. U. virens inocula were prepared from 7-day-old PSB-cultured P4, and 10 nM siRNA was added again in inocula before artificial inoculation. The control group was added with DEPC-treated ddH2O.
DNA polymorphism analysis
Primer pair Uv1261_SNPF/Uv1261_SNPR was used for amplifying the full-length DNA of UV_1261. To identify the evolutionary relationships among the examined U. virens isolates, primer pair UvSNP1_F/UvSNP1_R was used for amplifying a SNP-rich region in U. virens genome (Sun et al. 2013), and the obtained sequences were subjected to evolutionary analysis in MEGA5 using the Neighbor-Joining method with default parameters (Tamura et al. 2011). Primers are presented in Additional file 5: Table S2.
Sequence analysis and data processing
BLAST analysis was conducted at NCBI online (http://blast.ncbi.nlm.nih.gov/). Signal peptide prediction of UV_1261 was performed on SignalP 4.0 Server (http://www.cbs.dtu.dk/services/SignalP-4.0/) (Petersen et al. 2011). Sequence alignment was carried out with MultAlin (http://multalin.toulouse.inra.fr/multalin/multalin.html) (Corpet 1988). Conserved domain analysis was performed with Interpro search (http://www.ebi.ac.uk/interpro). Excel and SigmaPlot Version 10.0 were used for data processing. SPSS Version 21 was applied for statistical analysis.
Enhanced yellow fluorescent protein
Pathogen-associated molecular pattern
Potato sucrose agar
Potato sucrose broth
Rice false smut
Reactive oxygen species
Quantitative reverse transcription-polymerase chain reaction
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We thank Dr. YF Liu (Jiangsu Academy of Agricultural Sciences) for kindly providing the GFP-tagged U. virens isolate P4 and other U. virens isolates purified from RFS balls in Jiangsu Province. We thank Drs. DW Hu (Zhejiang University), CX Luo (Huazhong Agricultural University) and CQ Zang (Liaoning Academy of Agricultural Sciences) for providing U. virens isolates used for polymorphism analysis. We also thank Dr. WX Sun (China Agricultural University) for the courtesy of yeast strain YTK12 and pSUC2-derived plasmids. We thank Dr. JMJ Jeyakumar (Sichuan Agricultural University) for critical reading of this manuscript.
National Natural Science Foundation of China (grant no. 31501598 and 31772241) and Key Projects of Sichuan Provincial Education Department.
Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information files.
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The authors declare that they have no competing interests.
: Figure S1. Sequence alignment between PJ52_UV1261 and UV8b_UV1261. (TIF 3479 kb)
: Figure S2. a Mature protein sequence of UV_1261 (without signal peptide) was fused to eYFP and transiently expressed in leaves of N. benthamiana. Empty vector (EV) expressing only eYFP was set as a control. The construct 2 × RFP-NLS was co-expressed as a nucleus indicator. Scale bar, 10 μm. b Fluorescence intensity curves were drawn according to the direction of the red arrow in a. c Western blot with GFP antibody in N. benthamiana leaves expressing eYFP or UV_1261-eYFP. The band at 40 kDa indicates the UV_1261-eYFP fusion protein, while the band at 27 kDa presents free eYFP. d Flg22-induced ROS burst in N. benthamiana leaves transiently expressing UV_1261-eYFP or empty vector (CK). (TIF 1569 kb)
: Table S1. U. virens isolates used for DNA polymorphism analysis. (XLSX 10 kb)
: Figure S3. a Evolutionary relationships of 48 U. virens isolates from different rice production areas. Evolutionary analyses were conducted in MEGA5 using the Neighbor-Joining method with default parameters. The evolutionary distances were calculated using the Maximum Composite Likelihood method. Bootstrap test with 1000 replicates was performed. b Sequence alignment of UV_1261 among 49 U. virens isolates revealed that all the examined UV_1261 sequences were identical. Uv8b is the isolate that has a reference genome (Zhang et al. 2014). Information of other 48 isolates is presented in Additional file 3: Table S1. (TIF 9076 kb)
: Table S2. Primers used in this study. (XLSX 10 kb)
: Figure S4. Schematic diagram of vector Pzp-bar-Ex used for U. virens transformation. (TIF 123 kb)