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Kachewar et al. BMC Plant Biology (2019) 19:530 https://doi.org/10.1186/s12870-019-2079-1 RESEARCH ARTICLE Open Access Overexpression of OsPUB41, a Rice E3 ubiquitin ligase induced by cell wall degrading enzymes, enhances immune responses in Rice and Arabidopsis Neha Rajendra Kachewar1, Vishal Gupta1, Ashish Ranjan1,3, Hitendra Kumar Patel1 and Ramesh V. Sonti1,2* Abstract Background: Cell wall degrading enzymes (CWDEs) induce plant immune responses and E3 ubiquitin ligases are known to play important roles in regulating plant defenses. Expression of the rice E3 ubiquitin ligase, OsPUB41, is enhanced upon treatment of leaves with Xanthomonas oryzae pv. oryzae (Xoo) secreted CWDEs such as Cellulase and Lipase/Esterase. However, it is not reported to have a role in elicitation of immune responses. Results: Expression of the rice E3 ubiquitin ligase, OsPUB41, is induced when rice leaves are treated with either CWDEs, pathogen associated molecular patterns (PAMPs), damage associated molecular patterns (DAMPs) or pathogens. Overexpression of OsPUB41 leads to induction of callose deposition, enhanced tolerance to Xoo and Rhizoctonia solani infection in rice and Arabidopsis respectively. In rice, transient overexpression of OsPUB41 leads to enhanced expression of PR genes and SA as well as JA biosynthetic and response genes. However, in Arabidopsis, ectopic expression of OsPUB41 results in upregulation of only JA biosynthetic and response genes. Transient overexpression of either of the two biochemically inactive mutants (OsPUB41C40A and OsPUB41V51R) of OsPUB41 in rice and stable transgenics in Arabidopsis ectopically expressing OsPUB41C40A failed to elicit immune responses. This indicates that the E3 ligase activity of OsPUB41 protein is essential for induction of plant defense responses. Conclusion: The results presented here suggest that OsPUB41 is possibly involved in elicitation of CWDE triggered immune responses in rice. Keywords: Cell wall degrading enzymes, Damage associated molecular patterns, E3 ubiquitin ligase, Xoo, OsPUB41, Plant immunity and Rhizoctonia solani Background Plants have evolved very intricate and complex systems to cope with microbial infection. One of them is PAMPtriggered immunity (PTI), which is induced upon recognition of either conserved microbial molecules called pathogen-associated molecular patterns (PAMPs) or their own molecules released due to damage caused by the pathogen called damage-associated molecular patterns (DAMPs). Activation of PTI leads to various responses * Correspondence: sonti@ccmb.res.in 1 CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India 2 National Institute of Plant Genome Research, New Delhi 110067, India Full list of author information is available at the end of the article like callose deposition, production of reactive oxygen species, expression of defense genes, etc. [1]. Cell wall degrading enzymes (CWDEs) secreted by microbial pathogens have been long known to elicit plant defense responses such as production of phytoalexins, oxidative burst, strengthening of cell wall, etc. [2]. Endopolygalacturonic acid lyase, purified from Erwinia carotovora culture filtrates, has been shown to release oligosaccharides from soybean cell walls and thereby trigger phytoalexin accumulation in soybean [3]. Prior treatment of tobacco seedlings with CWDEs like pectate lyase or polygalacturonase from Erwinia carotovora subsp. carotovora induced resistance against subsequent E. c. subsp. carotovora infection [4]. However, the © The Author(s). 2019 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. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kachewar et al. BMC Plant Biology (2019) 19:530 Page 2 of 17 molecules involved in regulation of CWDEs induced plant innate immunity are not well studied. Xanthomonas oryzae pv. oryzae (Xoo), the bacterial blight pathogen of rice, secretes a battery of cell wall degrading enzymes (CWDE) such as lipase/esterase (LipA), cellulase (ClsA), xylanase (XynB), and cellobiosidase (CbsA) [5, 6]. Although they are important for virulence, these enzymes are double-edged swords as they induce rice defense responses such as callose deposition and programmed cell death. Also, prior treatment of rice leaves with any of these enzymes results in enhanced tolerance to subsequent Xoo infection [5]. It appears that CWDEs such as LipA act upon rice cell walls and release degradation products that act as DAMPs and elicit defense responses. In order to identify rice functions that may be involved in CWDE-induced defense responses, we had performed transcriptome analyses following treatment of rice leaves with purified ClsA [7] (12 h post-treatment with the enzyme) and LipA [12 h time-point [8] and 2 h time-point: GEO-ID: GSE53940]. In all of these analyses, the expression of OsPUB41, an E3 ubiquitin ligase gene (Class III U-box type), was found to be enhanced. In addition, at 2 h time-point OsPUB41 was the only E3 ligase, whose expression was significantly induced (> 1.5 fold up and p value ≤0.05) at this time point amongst 77 U-box genes annotated in rice genome. E3 ligases are known to be involved in regulating plant innate immune responses [9–12]. Earlier reports have indicated that the predicted protein contains an N-terminal U-box domain (~ 70 amino acids) and a conserved GKL domain (~ 100 amino acids with conserved glycine [G] and lysine [K]/arginine [R] residues as well as a leucine [L]-rich feature) near the C-terminus [11, 13]. OsPUB41 has been shown to be an active polyubiquitinating E3 ligase [13]. Here we report that transient overexpression of OsPUB41 in rice leaves results in induction of callose deposition and enhanced resistance against subsequent Xoo infection. Transient overexpression of OsPUB41 in rice induces expression of various JA and SA biosynthetic and response genes along with a set of Pathogenesis Related genes. Stable transgenics in Arabidopsis ectopically expressing OsPUB41 exhibited enhanced callose deposition, expression of various JA biosynthetic as well as response genes and enhanced tolerance to Rhizoctonia solani AGI-1A (R. solani) infection. In addition, overexpression of biochemically inactive OsPUB41 mutants (OsPUB41C40A and OsPUB41V51R) failed to elicit immune responses in rice and Arabidopsis. These results indicate that OsPUB41 might be a positive regulator of innate immunity and that the biochemical activity of OsPUB41 is necessary for elicitation of defense responses. Results Enhanced expression of OsPUB41 was observed following treatment with either CWDEs, elicitors or pathogens Previously, microarray analyses had been performed following treatment of rice leaves with either purified LipA [12 h time point [8] and 2 h time point, GEO-ID: GSE53940] or ClsA [7]. OsPUB41 was the only E3 ubiquitin ligase gene that was found to be upregulated in all of these treatments (Table 1). In addition, expression of OsPUB41 was found to be enhanced when rice leaves were infiltrated with commercially available CWDEs such as fungal cellulase, pectinase and xylanase. Interestingly, OsPUB41 expression was also induced upon treating rice with either DAMPs (eATP and sucrose) or PAMPs (Flg22 or LPS) (Additional file 1: Table S1). In addition, analysis of publicly available microarray data from GEO database revealed that the expression of OsPUB41 is enhanced when rice is infected by either a fungal (Magnaporthe grisea FR13 and Magnaporthe oryzae Guy11) or a bacterial (Xoo strains: PXO99A and PXO86) (Additional file 2: Table S2) pathogen. In addition, treatment of TN1 rice leaves with Xoo (strain BXO43; that we used in the experiment) also induced expression of OsPUB41 (Additional file 2: Table S2). Table 1 Expression of OsPUB41 is induced following treatment of rice leaves with various cell wall degrading enzymes Experimental parameters Microarray data b qPCR data c CWDEsa Lipase A (from Xoo) Cellulase A (from Xoo) Cellulase (from Trichoderma Xylanase (from Trichoderma Pectinase (from Aspergillus viride) viride) niger) Time point 2h 12 h 12 h 12 h 12 h Fold change in OsPUB41 expression 3.15 10.4 2.8 4.3 ± 1.16 2.9 ± 0.1 11 ± 4.6 12 h a Leaves of ten-fifteen-days-old Taichung Native-1 (TN-1) rice plants were pressure infiltrated, using a needleless syringe, with any one of the bacterial or fungal CWDEs b Relative fold change of OsPUB41 in microarray analysis observed using either Lipase A (GEO-ID: GSE53940, GSE49242) or Cellulase A (GSE8216) (p < 0.05) c Relative fold change of OsPUB41 (average value from three independent experiments, ± represents standard error) when treated with CWDEs as compared to mock treatment. OsActin was used as an internal control in qPCR for rice. Student’s two-tailed t-test for independent means was performed on delta Ct values to test for significance (p < 0.05) Kachewar et al. BMC Plant Biology (2019) 19:530 We analysed the OsPUB41 protein sequence using InterPro tool [14] which suggested that OsPUB41 has an N- terminal U-box domain (amino acid position 33 to 112) and an Armadillo-type fold (amino acid position 141–435) (Additional file 3: Fig. S1A, S1B) which are known to mediate ubiquitination and protein-protein interactions, respectively. Transient overexpression of OsPUB41 in rice leaves results in elicitation of callose deposition and enhanced tolerance to infection by Xoo Callose deposition is a marker of plant innate immune response. Purified preparations of CWDEs such as LipA, ClsA or CbsA induce callose deposition in rice leaves [5, 15]. OsPUB41 was transiently overexpressed in rice and the effect on callose deposition was assessed. For transient overexpression, an estradiol-inducible construct was used [16]. In the presence of inducer (Estradiol), the expression of OsPUB41 was induced 12–13 fold as compared to the uninduced state (DMSO) (Additional file 4: Fig. S2A). Immunoblotting was also performed to confirm expression of OsPUB41 in rice leaves (Additional file 4: Fig. S2B). Under conditions of OsPUB41 overexpression, there is a significant increase in the number of callose deposits (Fig. 1a, b). Estradiol by itself does not induce callose deposition in rice (Additional file 5: Table. S3). Prior treatment of rice leaves with a CWDE such as LipA or ClsA results in enhanced tolerance to subsequent Xoo infection [5]. Therefore, we checked whether overexpression of OsPUB41 would result in enhanced tolerance to Xoo infection. For this, midveins of leaves of 40–45-days-old rice plants were injected with Agrobacterium containing an estradiol-inducible OsPUB41 overexpression construct in the presence (induced) or absence (uninduced) of estradiol. Twelve hours later, these plants were infected with Xoo by pricking their midveins with a needle touched to a pellet of saturated Xoo culture. Lesion lengths were measured 12 days after infection. Under conditions of OsPUB41 expression, the lesion lengths were about 14 cm long while the lesion lengths were about 29 cm long in the absence of OsPUB41 overexpression. Similar size lesions (approximately 29 cm long) were observed in rice leaves that had been treated with Xoo without any prior treatment with Agrobacterium (Fig. 1c, d). Thus, overexpression of OsPUB41 leads to enhanced tolerance against subsequent Xoo infection in rice. Estradiol by itself does not affect Xoo infection in rice (Additional file 6: Table S4). Transient overexpression of OsPUB41 results in enhanced expression of Rice defense genes A number of JA biosynthetic and response genes were found to be upregulated 12 h post treatment of rice leaves with purified CWDEs like ClsA [7] or LipA [8]. Treatment Page 3 of 17 with LipA also led to an increase in the levels of (+)-7-isoJasmonoyl-L-isoleucine (JA-Ile), the bioactive form of JA [8]. We wanted to know if overexpression of OsPUB41 would lead to an increase in the level of expression of genes associated with either JA biosynthesis or response. Similar (to LipA or ClsA treated rice leaves) fold changes or enhancements in expression of JA biosynthetic as well as response genes were observed upon transient overexpression of OsPUB41 in rice leaves (Fig. 2a). We also found that overexpression of OsPUB41 induced the expression of genes associated with SA biosynthesis and response (Fig. 2b). Expression of rice defense genes like PR1a, PR1b, PR2, PR3, PR5 and PR9 was also induced by overexpression of OsPUB41 (Fig. 2c). Estradiol by itself neither affects expression of PR genes nor that of biosynthetic and response genes of JA and SA (Additional file 7: Table S5). Ectopic expression of OsPUB41 results in induction of callose deposition in transgenic Arabidopsis lines Transgenic Arabidopsis plants that exhibit estradiolinducible expression of OsPUB41 were generated. The expression of OsPUB41 was induced ~ 12 fold when the inducer (estradiol) was infiltrated into Arabidopsis leaves in comparison to the uninduced state (Additional file 8: Fig. S3). Induction of expression of OsPUB41 led to a significant increase in the number of callose deposits (Fig. 3a, b). Similar results were obtained in three transgenic Arabidopsis lines ectopically expressing OsPUB41 (Additional file 9: Table. S6). Estradiol by itself does not induce callose deposition (Additional file 5: Table S3). Hence, as observed in rice, expression of OsPUB41 enhances callose deposition in Arabidopsis. Ectopic expression of OsPUB41 results in enhanced expression of Arabidopsis genes involved in JA biosynthesis and response Treatment with either cellulase (Sigma) or exposure to a DAMP like AtPEP1 has been reported to induce the expression of JA biosynthetic and response genes [17, 18]. A number of JA and SA biosynthetic and response genes were found to be upregulated upon transient overexpression of OsPUB41 in rice. We wanted to know if ectopic expression of OsPUB41 leads to an increase in the level of expression of Arabidopsis genes associated with JA and SA biosynthesis or response. An increased expression of JA markers, biosynthetic as well as response genes was observed in a transgenic Arabidopsis line ectopically expressing OsPUB41 in an estradiol-inducible manner (Fig. 3c). A similar trend was observed in three independent transgenic Arabidopsis lines (Additional file 10: Table S7). In addition, the fold changes or enhancements observed in JA biosynthetic and response genes upon OsPUB41 expression in Arabidopsis were comparable to Kachewar et al. BMC Plant Biology Fig. 1 (See legend on next page.) (2019) 19:530 Page 4 of 17 Kachewar et al. BMC Plant Biology (2019) 19:530 Page 5 of 17 (See figure on previous page.) Fig. 1 Overexpression of OsPUB41 induces callose deposition and provides enhanced tolerance to Xoo infection in rice. Rice leaves were infiltrated with Agrobacterium LBA4404/pMDC7-OsPUB41 strain either with inducer (estradiol) or with DMSO (control). Twelve hours later, the leaves were stained with aniline blue and observed under an epifluorescence microscope. Bright spots in the images represent callose deposits (a). Scale bar represents 50 μm. The graph represents average number of callose deposits per field of view (0.075 mm2) from atleast ten leaves with six to eight different fields viewed per leaf in each experiment (b). Error bars represent standard error. Student’s two-tailed t-test for independent means was performed to test for significance (p < 0.05, represented by*). Similar results were obtained in three independent experiments. c. Xoo infections were carried out in midveins of leaves (n = 20–25) of 40-days-old TN-1 rice plants. The midveins were pre-injected with LBA4404/pMDC7-OsPUB41 with or without estradiol. After 12 h, these midveins were inoculated with Xoo (1–2 cm below the point where Agrobacterium was injected) by pricking with a needle dipped in a saturated Xoo culture. Yellowing represents bacterial blight lesions that were observed 12dpi. The graph represents average lesion lengths from at least twenty leaves in each experiment (d). Error bars represent standard error. Student’s two-tailed t-test for independent means was performed to test for significance (p < 0.05, ‘a’ and ‘b’ represent statistically different values). Similar results were obtained in three independent experiments those observed upon treating Arabidopsis leaves with either cellulase (Sigma) or AtPEP1. Ectopic expression of OsPUB41 did not affect the expression level of genes associated with either SA biosynthesis or response (Fig. 3d). Similar results were observed in three independent transgenic Arabidopsis lines (Additional file 10: Table S7). Estradiol (by itself) does not affect expression of JA or SA biosynthetic and response genes in Arabidopsis (Additional file 7: Table S5). Ectopic expression of OsPUB41 results in enhanced tolerance to Rhizoctonia solani AG1-IA infection in Arabidopsis Overexpression of OsPUB41 provides enhanced tolerance to subsequent Xoo infection in rice. We wanted to know whether ectopic expression of OsPUB41 would provide enhanced tolerance to microbial infection in Arabidopsis. Transgenic Arabidopsis lines expressing OsPUB41 were infected with either Rhizoctonia solani AG1-IA (R. solani, a fungal necroptroph) or Pseudomonas syringae pv. tomato DC3000 (Pst, a bacterial hemibiotroph). In the transgenic OsPUB41 Arabidopsis lines, induction of expression of OsPUB41 (by treatment with estradiol) led to enhanced tolerance to R. solani infection (Fig. 4a, b) as compared to uninduced control plants. As an additional control, wild type Arabidopsis (ecotype Col-0) was used (with and without estradiol) in the infection assay to rule out the possibility that estradiol could have an effect on the infection process. A scoring scale of zero to three based on fungal load was used for assessing the extent and severity of R. solani infection with zero indicating a high level of tolerance and three indicating a high level of susceptibility. A majority of wild type Arabidopsis plants (with and without estradiol) and the OsPUB41 transgenic plants (without inducer) exhibited scores of two or three. In contrast, a majority of OsPUB41 transgenic plants in which the expression of OsPUB41 had been induced exhibited a score of zero or one. Similar results were obtained in additional transgenic lines (Additional file 11: Table S8). Apart from qualitative analysis, the relative expression level of a fungal gene [18-28S ribosomal (r)DNA] as compared to a plant gene (AtUbq5) between induced (Estradiol) and uninduced (DMSO) samples indicated significantly less fungal load in OsPUB41 expressing lines. (Value ~ 1 indicates similar fungal load whereas a value < 1, indicates less fungal load) (Additional file 12: Fig. S4). Similar results were obtained in additional transgenic lines (Additional file 13: Table S9). This shows that OsPUB41 expression leads to enhanced tolerance to R. solani infection in Arabidopsis. Bacterial counts obtained from growth yield assays performed in Col-0 and transgenic Arabidopsis plants (with or without induction), post Pst infection were similar (p value > 0.05) (Additional file 14: Fig. S5). Similar results were obtained in additional transgenic lines (Additional file 15: Table S10). Expression of OsPUB41 had no significant effect on Pst infection in Arabidopsis. The C40A and V51R mutations of OsPUB41 affect the ability of the protein to elicit callose deposition and tolerance to Xoo infection in rice OsPUB41 mutant forms (OsPUB41C40A or OsPUB41V51R) were bacterially expressed, purified (Additional file 16: Fig. S6) and found to be biochemically inactive in an in vitro auto-ubiquitination assay in which OsPUB41 was active (Additional file 17: Fig. S7). Expression levels of OsPUB41 mutants (OsPUB41C40A or OsPUB41V51R) upon transient overexpression, as confirmed by qPCR (Additional file 4: Fig. S2A) and immunoblotting (Additional file 4: Fig. S2B) were found to be similar to that of OsPUB41. In Agrobacterium mediated transient transfer assays, induction of expression of the OsPUB41 mutants did not result in enhanced callose deposition in rice leaves (Fig. 5a). In contrast, induction of expression of wild type OsPUB41 led to enhanced callose deposition. Induction of expression of either OsPUB41C40A or OsPUB41V51R mutants did not result in enhanced tolerance to Xoo infection in rice leaves. Lesions of similar lengths were produced, irrespective of whether Kachewar et al. BMC Plant Biology (2019) 19:530 Page 6 of 17 Fig. 2 Transient overexpression of OsPUB41 leads to enhanced expression of rice defense genes. Transcript levels of JA (a) and SA (b) biosynthetic and response genes and of PR genes (c), were measured by qPCR upon overexpression of OsPUB41 in rice. AOS2: Allene oxide synthase2, AOC: Allene oxide cyclase, LOX2: Lipoxygenase2 and OPR2 and OPR4: 12-oxophytodienoate reductase2 and 4 (JA biosynthetic genes), JAZ8 and JAZ13: Jasmonate ZIM-Domain8 and 13 (JA response genes), PAL1 and PAL2: Phenylalanine ammonia lyase1 and 2 (SA biosynthetic genes), SGT1: SA glucosyltransferase1, NH1: Non-expresser of PR1 homolog1 (SA response genes), WRKY13: SA and JA response gene. OsActin was used as an internal control in qPCR. The graph represents average fold change values from three biological replicates. Student’s two-tailed t-test for independent means was performed on delta Ct values to test for significance (p < 0.05) expression of genes encoding OsPUB41C40A and OsPUB41V51R mutations were induced or not induced (Fig. 5b). In contrast, induction of expression of wild type OsPUB41 gene led to reduction in lesions caused by Xoo. The C40A mutation of OsPUB41 affects the ability of the protein to elicit callose deposition and tolerance to R. solani infection in Arabidopsis Transgenic Arabidopsis plants that exhibit estradiolinducible expression of OsPUB41C40A were generated. Kachewar et al. BMC Plant Biology Fig. 3 (See legend on next page.) (2019) 19:530 Page 7 of 17 Kachewar et al. BMC Plant Biology (2019) 19:530 Page 8 of 17 (See figure on previous page.) Fig. 3 Ectopic expression of OsPUB41 induces callose deposition, leads to enhanced expression of JA biosynthesis and response genes, but does not affect expression of SA biosynthesis and response genes in transgenic Arabidopsis lines. Leaves of thirty-days-old OsPUB41 transgenic Arabidopsis plants were infiltrated either with estradiol (inducer) or with DMSO (uninduced). After 12 h, leaves were stained with aniline blue and observed under an epifluorescence microscope. Bright spots in the images represent callose deposits (a). Scale bar represents 50 μm. The graph represents average number of callose deposits per field of view (0.075 mm2) from five-six leaves with ten different fields viewed per leaf in each experiment (b). Error bars represent standard error. Student’s two-tailed t-test for independent means was performed to test for significance (p < 0.05, represented by*). Similar results were obtained in three independent experiments (per transgenic line) and in three independent transgenic lines. Transcript levels of JA (c) and SA (d), biosynthetic and response genes, were measured upon ectopic expression of OsPUB41 in Arabidopsis. AOS: Allene Oxide Synthase (JA biosynthetic gene), PDF1.2a: Plant Defensin, VSP: Vegetative Storage Protein and JAZ: Jasmonate ZIM-Domain (JA response genes). SID2: SA Induction-Deficient 2 (SA biosynthetic gene), PAL2: Phenylalanine Ammonia-Lyase 2 (SA biosynthetic gene), NPR1: Nonexpresser of PR1 (SA response gene), PR1: Pathogenesis Related gene 1 (SA response gene) and PR5: Pathogenesis Related gene 5 (SA response gene). AtUbq5 was used as an internal control in qPCR. Three biological repeats were performed for each independent transgenic line. Similar results were obtained in three independent transgenic lines. Student’s two-tailed t-test for independent means was performed on delta Ct values to test for significance (p < 0.05) Expression level of OsPUB41C40A was found to be similar to that of OsPUB41 upon induction in transgenic Arabidopsis plants (Additional file 8: Fig. S3). Unlike transgenic plants expressing wild type OsPUB41, no enhancement in amount of callose deposition was observed in Arabidopsis transgenic lines expressing OsPUB41C40A (Fig. 6a). Similar results were obtained in additional transgenic lines ectopically expressing either OsPUB41 or OsPUB41C40A (Additional file 9: Table S6). Similarly, the level of R. solani infection was the same for OsPUB41C40A transgenic plants irrespective of whether or not the expression of the gene is induced. In contrast, the transgenic plants carrying wild type OsPUB41 showed a significant enhancement in tolerance to R. solani infection when expression of the transgene is induced (Fig. 6b). Similar results were obtained in additional transgenic lines ectopically expressing either OsPUB41 or OsPUB41C40A (Additional file 11: Table S8). Apart from qualitative analysis, the relative expression level of a fungal gene [18-28S ribosomal (r)DNA] as compared to a plant gene (AtUbq5) between induced (Estradiol) and uninduced (DMSO) samples indicated a similar fungal load in OsPUB41C40A expressing lines. (Value ~ 1 indicates similar fungal load whereas a value < 1 indicates less fungal load) (Additional file 12: Fig. S4). Similar results were obtained in additional transgenic lines (Additional file 13: Table S9). This shows that unlike for wild type OsPUB41, the expression of OsPUB41C40A does not lead to enhanced tolerance to R. solani infection in Arabidopsis. Fig. 4 Ectopic expression of OsPUB41 results in enhanced tolerance to R. solani infection in Arabidopsis. Fifteen-days-old Arabidopsis seedlings (Col-0 and OsPUB41 transgenics) were infected with R. solani, in either the presence or absence of the inducer (Estradiol) of OsPUB41 expression. Seven-dayspost infection, the seedlings were stained with Trypan Blue and imaged using a light microscope. A scale with scores ranging from 0 to 3 was used to assess the extent of fungal infection. Score 0 = no hyphae, 1 = few unconnected hyphae, 2 = sparse continuous network of hyphae and 3 = dense network of hyphae (a). Scale bar represents 50 μm. The graph represents the frequency (as a percentage) of a particular score from ten seedlings with forty different fields viewed per seedling in each experiment (b). Scores of 0, 1, 2 and 3 are represented in the graph by black, dark grey, light grey and white respectively. Similar results were observed in three independent experiments (per transgenic line) and in three independent transgenic lines. One-way ANOVA was used to test for significance, followed by Tukey-Kramer honestly significant difference test (p < 0.05, represented by*) Kachewar et al. BMC Plant Biology (2019) 19:530 Page 9 of 17 Fig. 5 OsPUB41 mutant forms are incapable of inducing callose deposition and tolerance to Xoo infection in rice. Callose deposition was assayed upon transient overexpression of either OsPUB41 or OsPUB41C40A or OsPUB41V51R. The graph represents average number of callose deposits per field of view (0.075 mm2) from ten leaves with six to eight different fields viewed per leaf in each experiment (a). Error bars represent standard error. Student’s two-tailed t-test for independent means was performed to test for significance (p < 0.05, represented by*). Similar results were obtained in three independent experiments. C40A and V51R represent OsPUB41C40A and OsPUB41V51R respectively. b. The bar represents average bacterial blight lesion length. Error bar represents standard error. Data was analyzed using the Student’s t-test for independent means (*indicates significant difference with p value < 0.05). Similar results were obtained in three independent experiments. C40A and V51R labels represent OsPUB41C40A and OsPUB41V51R respectively. Xoo label represents lesion lengths for leaves with Xoo infection without any prior treatment of Agrobacterium Fig. 6 OsPUB41C40A is incapable of eliciting callose deposition and tolerance to R. solani infection in Arabidopsis. The graph represents average number of callose deposits per field of view (0.075 mm2) from five-six leaves with ten different fields viewed per leaf in each experiment (a). Error bars represent standard error. Student’s two-tailed t-test for independent means was performed to test for significance (p < 0.05, represented by*). Similar results were obtained in three independent experiments with three independent transgenic lines. b. Transgenic Arabidopsis seedlings (carrying OsPUB41 or OsPUB41C40A) were infected with R. solani, either in the presence or absence of the inducer (Estradiol) of transgene expression. Infected seedlings were stained with Trypan Blue and imaged using a light microscope. A scale with scores ranging from 0 to 3 was used to assess the extent of fungal infection. Score 0 = no infection/hyphae, 1 = few unconnected hyphae, 2 = sparse continuous network of hyphae and 3 = dense network of hyphae. The graph represents the frequency (as a percentage) of a particular score from ten seedlings with forty different fields viewed per seedling in each experiment. Scores of 0, 1, 2 and 3 are represented in the graph by black, dark grey, light grey and white respectively. Seedlings from three independent transgenic lines were used for each experiment. A similar trend was observed in three independent experiments. One-way ANOVA was used to test for significance, followed by Tukey-Kramer honestly significant difference test (p < 0.05, represented by*). C40A label represents OsPUB41C40A Kachewar et al. BMC Plant Biology (2019) 19:530 Discussion CWDEs purified from Xoo induce defense responses in rice [5]. Very little information is available about rice functions that might be involved in elaboration of CWDE-induced immune responses. In microarray analysis of rice leaves treated with either one of two different CWDEs, namely LipA or ClsA, expression of one E3 ubiquitin ligase (OsPUB41) was consistently induced. OsPUB41 expression was also enhanced upon treatment with commercially available CWDEs (fungal cellulase, xylanase or pectinase). E3 ubiquitin ligases are known to involved in regulation of plant immune responses. OsPUB41 might participate in the signaling cascade activated upon cell wall damage. Hence, an enhancement in OsPUB41 expression is observed following cell wall damage. In addition to CWDEs, the expression of OsPUB41 was also induced upon exposure to pathogens. OsPUB41 expression was also induced upon exposure to several known PAMPs and DAMPs. The enhanced expression of OsPUB41 in presence of either a CWDE or an elicitor or a pathogen hinted towards the possibility that overexpression of this gene might mimic pathogen infection and result in elicitation independent enhancement of defense responses. Consistent with this possibility, overexpression of OsPUB41 was found to result in induction of callose deposition in rice and Arabidopsis. Transient overexpression of OsPUB41 imparted enhanced tolerance to Xoo infection in rice. In transgenic Arabidopsis plants ectopically expressing OsPUB41, enhanced tolerance to R. solani infection was observed. Thus, overexpression of OsPUB41 results in enhanced defense responses in rice and Arabidopsis. It appears that this protein may have a conserved role in elaboration of innate immune responses in rice (a monocot) and in Arabidopsis (a dicot). OsPUB41 has been earlier shown to be an E3 ligase. Mutants (OsPUB41C40A and OsPUB41V51R) of OsPUB41 that are defective in the E3 ligase activity of the protein failed to induce defense responses in rice and Arabidopsis. Hence, biochemical activity of OsPUB41 appears to be crucial for its role in induction of defense responses. The E3 ubiquitin ligases are known to play an important role in regulating plant immune signaling [9] by ubiquitination of their target proteins. The type of ubiquitination determines the fate of the substrate protein. Polyubiquitination through K48 marks the protein for degradation by 26S proteasome, whereas polyubiquitination with lysine linkages other than K48 and monoubiquitination regulate internalization and endocytotic trafficking of membrane receptors, histone modification, etc. [12]. E3 ubiquitin ligases can act as either negative or positive regulators of immune responses. For example, Rice SPL11 (Spotted Leaf11) is a negative regulator of cell death [19]. In Arabidopsis, the U-box E3 ligases Plant U-Box 12 (PUB12) and PUB13 attenuate PTI responses triggered upon recognition of flagellin [20]. In Chinese wild grapevine (Vitis pseudoreticulata), Page 10 of 17 EIRP1, a RING domain E3 ligase ubiquitinates VpWRKY11 (WRKY nuclear transcription factor) [21], which is a negative regulator of immune responses, and marks it for degradation. EIRP1 overexpression in Arabidopsis conferred enhanced tolerance to fungal and bacterial pathogens [21]. In rice, the RING-type E3 ligase, XA21 binding protein 3 (XB3) interacts with the receptor kinase protein XA21, which confers resistance against Xoo [22]. OsPUB15 has been shown to positively regulate plant innate immunity by interacting with rice receptor-like kinase PID2 [23]. Silencing of OsPUB44 resulted in suppression of peptidoglycan and chitin-induced immune responses suggesting a positive role for OsPUB44 in rice immunity. OsPUB44 has also been reported as a target of XopP (an Xoo effector protein) which suppresses peptidoglycan mediated immune responses. The XopP protein inhibits the E3 ligase activity of OsPUB44 [24]. At the moment, it is not clear whether the induction of immune responses following overexpression of OsPUB41 is due to the degradation of a negative regulator of innate immunity or whether it is a result of activation of a client protein through ubiquitination. Ectopic expression of OsPUB41 in Arabidopsis results in enhanced tolerance to R. solani, a necrotrophic fungal pathogen. Genes such as those encoding NADPH oxidases in Arabidopsis [25], OsWRKY80 [26] and chitinases [LOC_Os11g47510 [27]] in rice have been reported till date to provide enhanced tolerance to R. solani. Ectopic expression of OsPUB41 in Arabidopsis leads to enhanced expression of JA biosynthetic and response genes. Studies in Arabidopsis, tomato, and rice have shown that host resistance toward necrotrophs is conferred by ethylene and JA regulated signaling networks [28–30]. Also, it was observed that resistance against necrotrophic pathogens which is triggered by β-amino-butyric acid treatment is associated with induction of callose deposition [31]. We find that ectopic expression of OsPUB41 in Arabidopsis results in increased expression of certain JA biosynthesis and response genes and enhanced callose deposition. It is possible that the enhanced tolerance to R. solani in OsPUB41 expressing Arabidopsis plants is due to induction of callose deposition and other defense responses. OsPUB41 overexpression leads to enhanced tolerance to Xoo infection in rice. Exogenously applied SA (and SAmediated defenses) and JA (and JA-mediated defenses) were found to enhance tolerance to Xoo infection in rice [32–34]. In addition, overexpression of JA marker genes like AOS2, MYC2 or JAZ8 have been reported to modulate resistance to Xoo in rice [10, 34–36]. Overexpression of SA marker genes (OsSGT, OsPAL, OsNH1 or OsWRKY13) is known to impart resistance to Xoo infection in rice [37–40]. Transient overexpression of OsPUB41 in rice induces expression of markers of JA and SA signaling genes. In addition, overexpression of OsPUB41 triggers expression of PR genes (PR1a, PR1b,