Overexpression of OsPUB41, a Rice E3 ubiquitin ligase induced by cell wall degrading enzymes, enhances immune responses in Rice and Arabidopsis

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 withXanthomonas oryzaepv.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 ofOsPUB41leads to induction of callose deposition, enhanced tolerance to Xoo andRhizoctonia solaniinfection in rice and Arabidopsis respectively. In rice, transient overexpression ofOsPUB41leads to enhanced expression ofPRgenes and SA as well as JA biosynthetic and response genes. However, in Arabidopsis, ectopic expression ofOsPUB41results in upregulation of only JA biosynthetic and response genes. Transient overexpression of either of the two biochemically inactive mutants (OsPUB41C40AandOsPUB41V51R) ofOsPUB41in rice and stable transgenics in Arabidopsis ectopically expressingOsPUB41C40Afailed 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.

Plants have evolved very intricate and complex systems to cope with microbial infection. One of them is PAMP-triggered 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 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 fromErwinia carotovoraculture 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 fromErwinia carotovorasubsp.carotovorainduced resistance against subsequentE. c.subsp.carotovorainfection [4]。However, the molecules involved in regulation of CWDEs induced plant innate immunity are not well studied.

Xanthomonas oryzaepv.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]。看来,慢性消耗病如脂肪酶行动里克e 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 ofOsPUB41, an E3 ubiquitin ligase gene (Class III U-box type), was found to be enhanced. In addition, at 2 h time-pointOsPUB41was the only E3 ligase, whose expression was significantly induced (> 1.5 fold up andpvalue ≤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 [9,10,11,12]。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 ofOsPUB41in rice leaves results in induction of callose deposition and enhanced resistance against subsequent Xoo infection. Transient overexpression ofOsPUB41in rice induces expression of various JA and SA biosynthetic and response genes along with a set ofPathogenesis Relatedgenes. Stable transgenics in Arabidopsis ectopically expressingOsPUB41exhibited enhanced callose deposition, expression of various JA biosynthetic as well as response genes and enhanced tolerance toRhizoctonia solaniAGI-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.

Enhanced expression ofOsPUB41was 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]。OsPUB41was the only E3 ubiquitin ligase gene that was found to be upregulated in all of these treatments (Table1）.In addition, expression ofOsPUB41was found to be enhanced when rice leaves were infiltrated with commercially available CWDEs such as fungal cellulase, pectinase and xylanase.

Interestingly,OsPUB41expression was also induced upon treating rice with either DAMPs (eATP and sucrose) or PAMPs (Flg22 or LPS) (Additional file1: Table S1). In addition, analysis of publicly available microarray data from GEO database revealed that the expression ofOsPUB41is enhanced when rice is infected by either a fungal (Magnaporthe griseaFR13 andMagnaporthe oryzaeGuy11) or a bacterial (Xoo strains: PXO99A and PXO86) (Additional file2: Table S2) pathogen. In addition, treatment of TN1 rice leaves with Xoo (strain BXO43; that we used in the experiment) also induced expression ofOsPUB41(Additional file2: Table S2).

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 file3: Fig. S1A, S1B) which are known to mediate ubiquitination and protein-protein interactions, respectively.

Transient overexpression ofOsPUB41in 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]。OsPUB41was 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 ofOsPUB41was induced 12–13 fold as compared to the uninduced state (DMSO) (Additional file4: Fig. S2A). Immunoblotting was also performed to confirm expression ofOsPUB41in rice leaves (Additional file4: Fig. S2B). Under conditions ofOsPUB41overexpression, 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 file5: 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 ofOsPUB41would result in enhanced tolerance to Xoo infection. For this, midveins of leaves of 40–45-days-old rice plants were injected withAgrobacteriumcontaining an estradiol-inducibleOsPUB41overexpression 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 ofOsPUB41expression, the lesion lengths were about 14 cm long while the lesion lengths were about 29 cm long in the absence ofOsPUB41overexpression. Similar size lesions (approximately 29 cm long) were observed in rice leaves that had been treated with Xoo without any prior treatment withAgrobacterium(Fig.1c, d). Thus, overexpression ofOsPUB41leads to enhanced tolerance against subsequent Xoo infection in rice. Estradiol by itself does not affect Xoo infection in rice (Additional file6: Table S4).

Transient overexpression ofOsPUB41results 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 with LipA also led to an increase in the levels of (+)-7-iso-Jasmonoyl-L-isoleucine (JA-Ile), the bioactive form of JA [8]。We wanted to know if overexpression ofOsPUB41would 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 ofOsPUB41in rice leaves (Fig.2a). We also found that overexpression ofOsPUB41induced the expression of genes associated with SA biosynthesis and response (Fig.2b). Expression of rice defense genes likePR1a,PR1b,PR2,PR3,PR5andPR9was also induced by overexpression ofOsPUB41(Fig.2c). Estradiol by itself neither affects expression ofPR基因和生物合成和反应的基因of JA and SA (Additional file7: Table S5).

Ectopic expression ofOsPUB41results in induction of callose deposition in transgenic Arabidopsis lines

Transgenic Arabidopsis plants that exhibit estradiol-inducible expression ofOsPUB41were generated. The expression ofOsPUB41was induced ~ 12 fold when the inducer (estradiol) was infiltrated into Arabidopsis leaves in comparison to the uninduced state (Additional file8: Fig. S3). Induction of expression ofOsPUB41led to a significant increase in the number of callose deposits (Fig.3a, b). Similar results were obtained in three transgenic Arabidopsis lines ectopically expressingOsPUB41(Additional file9: Table. S6). Estradiol by itself does not induce callose deposition (Additional file5: Table S3). Hence, as observed in rice, expression ofOsPUB41enhances callose deposition in Arabidopsis.

Ectopic expression ofOsPUB41results 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 ofOsPUB41in rice. We wanted to know if ectopic expression ofOsPUB41leads 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 expressingOsPUB41in an estradiol-inducible manner (Fig.3c). A similar trend was observed in three independent transgenic Arabidopsis lines (Additional file10: Table S7). In addition, the fold changes or enhancements observed in JA biosynthetic and response genes uponOsPUB41expression in Arabidopsis were comparable to those observed upon treating Arabidopsis leaves with either cellulase (Sigma) or AtPEP1. Ectopic expression ofOsPUB41did 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 file10: Table S7). Estradiol (by itself) does not affect expression of JA or SA biosynthetic and response genes in Arabidopsis (Additional file7: Table S5).

Ectopic expression ofOsPUB41results in enhanced tolerance toRhizoctonia solaniAG1-IA infection in Arabidopsis

Overexpression ofOsPUB41provides enhanced tolerance to subsequent Xoo infection in rice. We wanted to know whether ectopic expression ofOsPUB41would provide enhanced tolerance to microbial infection in Arabidopsis. Transgenic Arabidopsis lines expressingOsPUB41were infected with eitherRhizoctonia solaniAG1-IA (R. solani, a fungal necroptroph) orPseudomonas syringaepv.tomatoDC3000 (Pst, a bacterial hemibiotroph).

In the transgenicOsPUB41Arabidopsis lines, induction of expression ofOsPUB41(by treatment with estradiol) led to enhanced tolerance toR. solaniinfection (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 ofR. solaniinfection 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 theOsPUB41transgenic plants (without inducer) exhibited scores of two or three. In contrast, a majority ofOsPUB41transgenic plants in which the expression ofOsPUB41had been induced exhibited a score of zero or one. Similar results were obtained in additional transgenic lines (Additional file11: 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 inOsPUB41表达。(价值~ 1表明类似的乐趣gal load whereas a value < 1, indicates less fungal load) (Additional file12: Fig. S4). Similar results were obtained in additional transgenic lines (Additional file13: Table S9). This shows thatOsPUB41expression leads to enhanced tolerance toR. solaniinfection 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 (pvalue > 0.05) (Additional file14: Fig. S5). Similar results were obtained in additional transgenic lines (Additional file15: Table S10). Expression ofOsPUB41had 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 (OsPUB41C40AorOsPUB41V51R) were bacterially expressed, purified (Additional file16: Fig. S6) and found to be biochemically inactive in an in vitro auto-ubiquitination assay in which OsPUB41 was active (Additional file17: Fig. S7). Expression levels ofOsPUB41mutants (OsPUB41C40AorOsPUB41V51R) upon transient overexpression, as confirmed by qPCR (Additional file4: Fig. S2A) and immunoblotting (Additional file4: Fig. S2B) were found to be similar to that ofOsPUB41. InAgrobacteriummediated transient transfer assays, induction of expression of theOsPUB41mutants did not result in enhanced callose deposition in rice leaves (Fig.5a). In contrast, induction of expression of wild typeOsPUB41led to enhanced callose deposition. Induction of expression of eitherOsPUB41C40AorOsPUB41V51Rmutants did not result in enhanced tolerance to Xoo infection in rice leaves. Lesions of similar lengths were produced, irrespective of whether expression of genes encodingOsPUB41C40AandOsPUB41V51Rmutations were induced or not induced (Fig.5b). In contrast, induction of expression of wild typeOsPUB41gene 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 toR. solaniinfection in Arabidopsis

Transgenic Arabidopsis plants that exhibit estradiol-inducible expression ofOsPUB41C40Awere generated. Expression level ofOsPUB41C40Awas found to be similar to that ofOsPUB41upon induction in transgenic Arabidopsis plants (Additional file8: Fig. S3). Unlike transgenic plants expressing wild typeOsPUB41, no enhancement in amount of callose deposition was observed in Arabidopsis transgenic lines expressingOsPUB41C40A(Fig.6a). Similar results were obtained in additional transgenic lines ectopically expressing eitherOsPUB41orOsPUB41C40A(Additional file9: Table S6). Similarly, the level ofR. solaniinfection was the same forOsPUB41C40Atransgenic plants irrespective of whether or not the expression of the gene is induced. In contrast, the transgenic plants carrying wild typeOsPUB41showed a significant enhancement in tolerance toR. solaniinfection when expression of the transgene is induced (Fig.6b). Similar results were obtained in additional transgenic lines ectopically expressing eitherOsPUB41orOsPUB41C40A(Additional file11: 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 inOsPUB41C40A表达。(价值~ 1表明类似的乐趣gal load whereas a value < 1 indicates less fungal load) (Additional file12: Fig. S4). Similar results were obtained in additional transgenic lines (Additional file13: Table S9). This shows that unlike for wild typeOsPUB41, the expression ofOsPUB41C40Adoes not lead to enhanced tolerance toR. solaniinfection in Arabidopsis.

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.OsPUB41expression 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.OsPUB41might participate in the signaling cascade activated upon cell wall damage. Hence, an enhancement inOsPUB41expression is observed following cell wall damage. In addition to CWDEs, the expression ofOsPUB41was also induced upon exposure to pathogens.OsPUB41expression was also induced upon exposure to several known PAMPs and DAMPs. The enhanced expression ofOsPUB41in 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 ofOsPUB41was found to result in induction of callose deposition in rice and Arabidopsis. Transient overexpression ofOsPUB41imparted enhanced tolerance to Xoo infection in rice. In transgenic Arabidopsis plants ectopically expressingOsPUB41, enhanced tolerance toR. solaniinfection was observed. Thus, overexpression ofOsPUB41结果在大米和增强防御反应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]。InArabidopsis, 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), 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 inArabidopsisconferred 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 ofOsPUB41is 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 ofOsPUB41in Arabidopsis results in enhanced tolerance toR. 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 toR. solani. Ectopic expression ofOsPUB41in 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 [28,29,30]。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 ofOsPUB41in Arabidopsis results in increased expression of certain JA biosynthesis and response genes and enhanced callose deposition. It is possible that the enhanced tolerance toR. solaniinOsPUB41expressing Arabidopsis plants is due to induction of callose deposition and other defense responses.

OsPUB41overexpression leads to enhanced tolerance to Xoo infection in rice. Exogenously applied SA (and SA-mediated defenses) and JA (and JA-mediated defenses) were found to enhance tolerance to Xoo infection in rice [32,33,34]。In addition, overexpression of JA marker genes likeAOS2,MYC2orJAZ8have been reported to modulate resistance to Xoo in rice [10,34,35,36]。Overexpression of SA marker genes (OsSGT,OsPAL,OsNH1orOsWRKY13) is known to impart resistance to Xoo infection in rice [37,38,39,40]。Transient overexpression ofOsPUB41in rice induces expression of markers of JA and SA signaling genes. In addition, overexpression ofOsPUB41triggers expression ofPRgenes (PR1a,PR1b,PR2,PR3,PR5andPR9) in rice. Induction ofPRgenes is reported to be associated with enhanced tolerance to pathogen infection [41,42]。Thus, the enhanced tolerance to Xoo infection which is observed uponOsPUB41overexpression might arise due to enhancement in expression of rice genes associated with JA and SA biosynthesis and response and genes from PR family.OsPUB41overexpression leads to enhanced tolerance to Xoo infection in rice but does not provide enhanced tolerance to Pst infection in Arabidopsis. Coronatine (a phytotoxin and a potent virulence factor of Pst) has been reported to suppress induction of callose deposition in Arabidopsis during infection [43]。Hence, although expression ofOsPUB41induces callose deposition, it may not provide enhanced tolerance to Pst infection in Arabidopsis. Pst is a hemibiotroph which during infection activates the JA pathway in Arabidopsis [44]。SA and JA are known to be antagonistic to each other in Arabidopsis [45,46]。Hence, by inappropriate activation of JA pathway, Pst suppresses SA pathway and SA mediated defense responses. Enhanced expression of SA responsive genes likeNPR1andPRgenes (PR1,PR2andPR5) is associated with resistance to Pst infection in Arabidopsis [47]。In addition, an alteration in expression of SA biosynthetic genes likeSID2andPAL(PAL1-PAL4genes) affect disease (caused by Pst) outcome in Arabidopsis [48,49]。OsPUB41expression in Arabidopsis induces expression of JA marker genes but has no effect on expression of SA marker genes (biosynthetic as well as response genes). It is possible that since the expression of SA related genes (biosynthetic and response genes) remained unaffected in Arabidopsis uponOsPUB41expression, neither resistance nor enhanced susceptibility to Pst infection was observed. Therefore, it is possible, that although both Xoo and Pst are hemibiotrophic in nature,OsPUB41expression provides enhanced tolerance to Xoo in rice but not to Pst in Arabidopsis.

There are certain differences in hormonal pathways in rice and Arabidopsis. For example: AtPAD4 (Phytoalexin deficient 4) is SA responsive and has a positive feedback/regulation with respect to SA. However, its orthologue, OsPAD4 interacts with both JA and SA signaling in rice [50]。In addition, role of AtEDS1 (Enhanced disease susceptibility) in plant (Arabidopsis) defense depends on SA signaling. However, role of OsEDS1, rice orthologue of AtEDS1, in plant (rice) defense depends on JA signaling. Expression of AtEDS1 in oseds1 (OsEDS1 knockout rice plants) partially complements oseds1 defense phenotype [51]。Apart from known commonalities, there exist quite a few subtle differences in the regulation and execution of defense responses in rice and Arabidopsis (monocots and dicots). Hence, it is possible thatOsPUB41induces expression of genes differently in rice and in Arabidopsis.

In summary,OsPUB41expression is induced following treatment of rice leaves with various CWDEs. Also, overexpression ofOsPUB41leads to induction of plant defense responses. This suggests thatOsPUB41might be involved in elaboration of CWDE-induced plant immune responses. At this point, we do not know whetherOsPUB41is essential for elicitation of rice immune responses following treatment with CWDEs. To address this issue,OsPUB41knockdown or knockout rice plants need to be generated using either RNAi or genome editing and their phenotypes need to be assessed following treatment with CWDEs.

OsPUB41might be a positive regulator of innate immunity that participates in PAMP and DAMP triggered signaling pathways.

Plant materials and growth conditions

Ten-fifteen-days-old seedlings of greenhouse grown bacterial blight susceptible rice cultivar, Taichung Native-1 (TN-1) [procured from Indian Institute of Rice Research (IIRR) and IIRR procured TN-1 from International Rice Research Institute (original source)], were used for qPCR and callose deposition. Forty-days-old, greenhouse-grown TN-1 plants were used for Xoo infection assays. TheArabidopsis thaliana(Arabidopsis) Columbia ecotype (Col-0) was used as wild type and for generating transgenic plants [Col-0 seeds from laboratory stock were used by the authors for all experiments. Seeds for maintaining laboratory stock were initially procured from Dr. Imran Siddiqi’s Lab (CCMB, India) who had originally procured them from TAIR (original source)]. Arabidopsis plants were grown and maintained as described earlier [18]。

Generation of plant expression plasmids (pMDC7-OsPUB41, pMDC7-OsPUB41C40A and pMDC7-OsPUB41V51R) using gateway cloning

TheOsPUB41gene (LOC_Os03g13740) encodes a CDS of length 1338 bps. The cDNA was prepared using Superscript III reverse transcriptase (Invitrogen) from total RNA isolated from LipA treated TN-1 rice leaves (2 h post-treatment) as described previously [8]。TheOsPUB41gene was cloned into the inducible plant expression vector, pMDC7, by Gateway cloning as per the manufacturer’s instructions (Invitrogen). Entry and destination constructs (pENTR-OsPUB41 and pMDC7-OsPUB41, respectively) were generated in this process. Entry constructs for two OsPUB41 mutants (pENTR-OsPUB41C40A and pENTR-OsPUB41V51R) containing substitutions (corresponding to amino acid residues) either at 40th (C to A) or 51st (V to R) position were constructed using Quick change site-directed mutagenesis kit (Stratagene), primers with altered codons (Additional file18: Table S11) and pENTR-OsPUB41 (as a template). From entry constructs of OsPUB41 mutants, destination constructs: pMDC7-OsPUB41C40A and pMDC7-OsPUB41V51R were obtained. In pMDC7, gene expression is under the control of the 17-β-estradiol inducible XVE promoter. These plant expression vectors containing eitherOsPUB41or its mutant forms were transformed intoAgrobacterium tumefaciensLBA4404 strain by electroporation and selected on medium containing appropriate antibiotics (Additional file19: Table S12). The LBA4404/pMDC7-OsPUB41, LBA4404/pMDC7-OsPUB41C40A and LBA4404/pMDC7-OsPUB41V51R clones were confirmed by colony PCR and subsequent sequencing of the PCR amplicons using vector specific primers (Additional file18: Table S11).

Generation of clones for bacterial expression ofOsPUB41and its mutant forms (OsPUB41C40AandOsPUB41V51R)

OsPUB41,OsPUB41C40AandOsPUB41V51Rwere amplified using pENTR-OsPUB41, pENTR-OsPUB41C40A and pENTR-OsPUB41V51R as templates respectively with KpnIF and Kpn1RNS primers (Additional file18: Table S11). Modified pETM40 (MpETM40) vector and the amplicons were digested with KpnI. The digested vector was treated with Antarctic phosphatase and ligated with either of the aforementioned KpnI digested amplicons. The ligation mixture was used for transformingE. coliDH5α cells. Clones were selected using appropriate antibiotics (Additional file19: Table S12) and subsequently confirmed by PCR and sequencing using MBPF and KpnIRNS primers (Additional file18: Table S11). MpETM40-OsPUB41, MpETM40-OsPUB41C40A and MpETM40-OsPUB41V51R were subsequently transformed intoE. coliBL21-DE3 individually for expression of the respective proteins. TheE. coliBL21-DE3 clones were further selected using appropriate antibiotics (Additional file19: Table S12) and subsequently confirmed by PCR and sequencing using MBPF and KpnIRNS primers (Additional file18: Table S11).

Generation of transgenic Arabidopsis plants

Arabidopsis Col-0 (wild type) was used to generate transgenic lines expressing eitherOsPUB41orOsPUB41C40A(mutant form ofOsPUB41) by floral dip method of transformation [52] usingAgrobacterium菌株LBA4404 pMDC7-OsPUB41或pMDC7 -OsPUB41C40A construct. During germination, the transformants were selected using hygromycin (25μgml^− 1）.Putative transgenic plants (hygromycin resistant T₁generation) were further confirmed by direct PCR (Terra PCR direct polymerase kit, Clontech) using leaf tissue and sequencing of the amplified product. Plants from three independent lines expressing eitherOsPUB41orOsPUB41C40A(genotype confirmed, T₂generation), were used in all experiments.

Analyses of publicly available microarray data from GEO database

The dotCEL files (.CEL) for LipA (GEO-ID: GSE53940 and GSE49242), ClsA (GEO-ID: GSE8216),Magnaporthe griseaFR13 (GEO-ID: GSE7256),Magnaporthe oryzaeGuy11 (GEO-ID: GSE18361),PXO99AandPXO86(GEO-ID: GSE36272) were downloaded from GEO (https://www.ncbi.nlm.nih.gov/geo/）.PLIER normalized dotCHP (.CHP) files were generated for these dotCEL files using Affymetrix Expression Console software (ThermoFisher Scientific). Using the Affymetrix Transcriptome Analysis Console these PLIER normalized dotCHP files were analysed to obtain relative fold change values for treated versus mock. Only relative fold change values forOsPUB41withpvalue < 0.05 have been tabulated.

Treatment of Rice leaves with CWDEs, elicitors or Xoo pathogen

Ten-fifteen-days-old, TN-1 plants were infiltrated with one of the following:

Xylanase, Pectinase, Cellulase (Sigma, 2.35 units each, mock: water), Sucrose (Sigma, 1 mM, mock: water), ATP (Calbiochem, 1 mM, mock: water), Flg22 (Genscript, 1 mM, mock: water) lipopolysaccharide [LPS purified from Xoo, 100 μg ml^− 1; method for LPS purification is as described [53], mock: water] and Xoo (BXO43 strain, resuspended in water, mock: water). Twelve hours post treatment, rice leaves were harvested for q-RTPCR assays.

Callose deposition assay

Callose deposition in rice was assayed upon transient overexpression ofOsPUB41or mutated forms ofOsPUB41(C40A and V51R).Agrobacteriumstrains containing either pMDC7-OsPUB41, pMDC7-OsPUB41C40A or pMDC7-OsPUB41V51R were grown, induced (by Acetosyringone) and infiltrated into rice leaves, either with the inducer (Estradiol) or without the inducer (DMSO) as previously described [18]。Twelve hours post infiltration these rice leaves were cut, processed, stained (with aniline blue) and viewed under an epifluorescence microscope, as previously described [18]。

Callose deposition assays were also performed in stable transgenic Arabidopsis lines expressing eitherOsPUB41orOsPUB41C40Aunder a 17–β–estradiol inducible system. Either inducer (Estradiol) or mock (DMSO) solution was infiltrated in the rosette stage leaves of T₂generation ofOsPUB41orOsPUB41C40Aexpressing plants using a needleless 1 ml syringe. Twelve hours post-infiltration, leaves were collected, processed and the assay for visualization of callose deposition was performed as mentioned above for rice leaves.

Xoo infection assay in rice

OsPUB41or its mutant forms were transiently overexpressed in midveins of the leaves of 40-days-old rice plants using either LBA4404/pMDC7-OsPUB41 or LBA4404/pMDC7-OsPUB41C40A or LBA4404/pMDC7-OsPUB41V51R cultures as described earlier [18]。Twelve hours later, the midveins of the leaves were infected with wild type Xoo (BXO43 strain) by pricking with a needle touched to a pellet of saturated culture. Lesion lengths were measured on the twelfth day post-infection.

Western-blotting

Leaves of fourteen-days-old TN-1 rice seedlings were infiltrated withAgrobacteriumstrains containing either pMDC7-OsPUB41, pMDC7-OsPUB41C40A or pMDC7-OsPUB41V51R constructs with or without estradiol. Infiltrated leaf samples were collected after 12 h and ground in liquid nitrogen followed by homogenization in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 250 mM Mannitol, 5 mM EDTA, 10% glycerol, 1 mM DTT, 1% Triton X-100, 1 mM PMSF and 1 mM NaF) [54]。离心后(15000克15分钟4°C),equal amounts of isolated protein supernatants were separated using two 15% SDS-PAGE gels. Each sample was split into two: first half was loaded in Gel 1 and second half of the same sample was loaded in Gel2. The OsPUB41 protein and its mutant forms were detected by Western blot analysis (Gel1 was used for this purpose) using polyclonal rabbit anti-OsPUB41Pep3 antibody [1: 1000 dilution, Genscript generated Ab using a peptide (Pep3; sequence: VAESAARRGAAGRAC) of OsPUB41]. Rabbit polyclonal Histone (H3, Abcam) antibody (1: 50,000) was used to detect Histone (loading control) in these samples (Gel2 was used for this purpose). HRP conjugated anti-rabbit secondary antibody (Abcam) was used and the protein bands were viewed using Luminata Forte HRP substrate (Millipore). Chemiluminescence imaging system with Chemi-capt 5000 software (version 12.8; Vilber Lourmat) was used for capturing the signal. Induced and uninduced (forOsPUB41expression) leaf tissues of Arabidopsis were processed in a similar manner.

For purified proteins (OsPUB41, OsPUB41C40A, OsPUB41V51R and MBP), immunoblotting was performed using mouse monoclonal anti-polyhistidine alkaline phosphatase antibody (1: 4000 dilution, Sigma). Approximately 100 ul of chromogenic substrate (Sigma, 66 μl NBT stock + 33 μl BCIP) in 10 ml of alkaline phosphatase buffer (100 mM NaCl, 5 mM MgCl₂and 100 mM Tris-Cl, pH 9.5) was added to the blot and incubated with gentle shaking. The bands appeared in 1–2 min and the reaction was stopped by washing the blot with water.

Rhizoctonia solaniAG1-IA infection in Arabidopsis seedlings

Arabidopsis seedlings (Col-0,OsPUB41andOsPUB41C40A) were grown for 15 days on vertically positioned agar plates containing ½ MS with inducer (Estradiol) or without the inducer (DMSO).R. solaniinfection was carried out as described previously [55]。Seven dpi, seedlings were washed twice with sterile water to remove superficially growing fungus and stained, for 1 min, with Trypan Blue solution (10 ml Lactic acid, 10 ml glycerol, 10 ml water, 10 g phenol, 10 g Trypan Blue) diluted in 96% ethanol in 1: 2 ratio. After three washes with destaining solution (10 ml Lactic acid, 10 ml glycerol, 10 ml MQ, 10 g phenol, diluted in 96% ethanol in 1: 2 ratio), seedlings were observed under the light microscope (10X objective).

Quantification ofR. solaniDNA from infected Arabidopsis leaves by real-time PCR

For fungal load determination, DNA was isolated from Arabidopsis seedlings (Col-0,OsPUB41andOsPUB41C40A), 7 dpi using the CTAB method [56]。Two ng of total DNA from each infected plant tissue sample was used for qPCR. UBQ5F and UBQ5R (plant specific) and Rs1F and Rs2R (fungus specific) primers (Additional file18: Table S11) [57] were used for qPCR performed on the Applied Biosystems ViiA7 Real-Time PCR System, using PowerSYBR Green/ROX Master Mix (ThermoFisher Scientific). The relative expression level of the fungal gene as compared to the plant gene between induced (Estradiol) and uninduced (DMSO) samples was calculated using the 2^(−ΔΔCt)method [58]。

Quantitative real time PCR (qRT-PCR)

Total RNA from Arabidopsis and rice leaves was isolated using TRIzol Reagent (ThermoFisher Scientific). After DNaseI treatment (NEB, according to manufacturer’s instructions), cDNA was synthesized with 1 μg of total RNA by Oligo (dT)-primed reverse transcription using EcoDry kit (Clontech, Takara, USA). qRT-PCR was performed on the Applied Biosystems ViiA 7 Real-Time PCR System using PowerSYBR Green/ROX Master Mix (ThermoFisher Scientific).OsActinandAtUbq5were used as internal controls for rice and Arabidopsis respectively. The primers used for qPCR have been listed in (Additional file18: Table S11). The relative expression of various genes between induced (Estradiol) and uninduced (DMSO) samples was calculated using the 2^(−ΔΔCt)method [58]。

Pseudomonas syringaepv.tomatoDC3000 (Pst) infection assay in Arabidopsis plants

Cultures of virulent Pst were grown to mid to late log phase in LB supplemented with rifampicin (50 μg ml^− 1) at 28 °C for 12 h before inoculation. Fully expanded leaves of wild-type and transgenic Arabidopsis plants were pressure infiltrated with either Pst (OD₆₀₀of 0.02) suspended in 10 mM MgCl₂with estradiol (inducer) or DMSO (control; uninduced) using a needleless syringe into three leaves per plant [59]。在每个实验中,每三个植物使用time point per condition (induced or uninduced) for each transgenic line. Bacterial growth assays were performed at 0 and 48 h post infection (hpi) to determine disease progression in the plants. Leaves were surface sterilised with 70% (v/v) ethanol and then washed in sterile water for 1 min. Each leaf was placed in 500 μl of 10 mM MgCl₂solution and crushed to acquire the bacteria. The resulting solution was serial diluted and 10 μl of each dilution was spotted on LB plates containing 50 μg ml^− 1rifampicin. The plates were incubated at 28 °C for 48 h prior to counting of the colonies.

Overexpression and purification of OsPUB41 and its mutant forms

Single colonies of selected BL21-DE3 clones containing either MpETM40-OsPUB41 or MpETM40-OsPUB41C40A or MpETM40-OsPUB41V51R or MpetM40 (empty vector), were inoculated into 5 ml LB medium containing 100 μg ml^− 1Kanamycin and grown overnight (37 °C, 200 rpm). Further, 300 ml LB medium containing 100 μg ml^− 1Kanamycin was inoculated with 1% inoculum of the overnight grown culture. The cultures were grown until mid-log phase (OD₆₀₀~ 0.4–0.6). After addition of IPTG (inducer: 1 mM), the cultures were further incubated (37 °C, 200 rpm) for 4–6 h. At the same time, uninduced cultures were also maintained. One ml of each culture was pelleted and used for SDS-PAGE analysis. The cultures were centrifuged (8000 rpm, 4 °C, 10 min). The cell pellet from the remaining 299 ml culture ofE. coliexpressing 6X-His-tagged recombinant protein was resuspended in 10 ml of Resuspension Buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl) containing 1 mM PMSF. The cells were disrupted by sonication on ice and centrifuged (12,000 g, 30 min, 4 °C). The supernatants were added to TALON beads pre-equilibrated with Resuspension Buffer. It was then kept overnight at 4 °C on a low speed rocker. Columns with these TALON beads were prepared in 50 ml falcons. The column was washed thrice with 50 ml of Wash Buffer-1 (50 mM Tris-HCl (pH 8), 150 mM NaCl, 10 mM Imidazole). After 3 washes with Wash Buffer-2 (50 mM Tris-HCl (pH 8), 500 mM NaCl), proteins were eluted using Elution Buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 100 mM Imidazole). The purified proteins were aliquoted and stored at − 80 °C until further use. OsPUB41 protein and its mutant forms have an N-terminal MBP tag and a C-terminal 6X-His tag. The empty vector (MpETM40), expresses MBP protein with a C-terminal 6X-His tag.

In vitro self-ubiquitination assay

Ubiquitination reactions of 100ul each containing either of the purifiedE. coliexpressed proteins; OsPUB41, OsPUB41C40A, OsPUB41V51R and MBP (10 uM each) along with ubiquitination reaction buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 1 mM dithiothreitol (DTT), 2 mM ATP), human E1, E2 UbcH5A (R&D Biosciences) and His-tagged penta-ubiquitin were incubated for 4 h at 37 °C. The reaction was stopped by the addition of SDS sample buffer. After boiling for 10 min, the samples were separated by 15% SDS-PAGE and subjected to immunoblotting using either of the following antibodies: polyclonal rabbit anti-MBP antibody (1: 2000 dilution, Abcam) or monoclonal mouse anti-polyHis antibody conjugated to alkaline phosphatase (1: 4000 dilution, Sigma) or mouse monoclonal anti-ubiquitin antibody (1: 1000 dilution, Santa Cruz Biotechnology). Donkey polyclonal antibody to rabbit IgG (1: 50,000 dilution, Abcam) or goat polyclonal antibody to mouse IgG (1: 2000 dilution, Abcam), conjugated to HRP was used as secondary antibody (1 h, at room temperature). Anti-His probed blot was developed as described above using a chromogenic substrate (NBT and BCIP).

Statistics

Unless mentioned, all experiments were performed atleast in three biological replicates. All experiments in Arabidopsis were reproduced in three independent transgenic lines. Statistical analysis was performed using the unpaired students t–test for independent means wherever necessary. One-way ANOVA followed by the Tukey-Kramer honestly significant difference test was used for analysing qPCR and scoring data forR. solaniinfection in Arabidopsis.

All data generated or analyzed during this study are included in this published article (and its additional files). Any material generated during the current study is available from the corresponding author on reasonable request.

CbsA:

cellobiosidase

ClsA:

Cellulase A

CWDE:

cell wall degrading enzyme

DAMP:

Damage associated molecular patterns

DMSO:

dimethyl sulfoxide

dpi:

days post infection or infiltration

Est:

17-β-Estradiol

hr.:

hour

JA:

Jasmonic acid

JA-Ile:

(+)-7-iso-Jasmonoyl-L-isoleucine

LipA:

Lipase/esterase A

LPS:

lipopolysaccharide

MBP:

Maltose binding protein

MS:

Murashige and Skoog’s

MSA:

multiple sequence alignment

PAMPs:

pathogen-associated molecular patterns

PR:

Pathogenesis Related

Pst:

Pseudomonas syringaepv.tomatoDC3000

PTI:

PAMP-triggered immunity

PUB:

Plant U-Box

R. solani:

Rhizoctonia solaniAGI-1A

ROS:

Reactive oxygen species

SA:

Salicylic acid

TN-1:

Taichung Native-1

Xoo:

Xanthomonas oryzaepv.oryzae

XynB:

xylanase

We acknowledge Dr. Gopaljee Jha, Dr. Subhadeep Chatterjee, Dr. Imran Siddiqi, Dr. Veena Parnaik and Dr. Ueli Grossniklaus for providingR. solani, Pst strains, MpETM40 vector, His-tagged penta-ubiquitin and pMDC7 vector respectively.

NRK, acknowledges an INSPIRE fellowship from the Department of Science and Technology (DST), Government of India. VG was supported by a post-doctoral fellowship from the Department of Biotechnology, Government of India. This work was supported by the XII^thfive year plan project, Plant-Microbe and Soil Interactions (BSC0117) of the Council of Scientific and Industrial Research. RVS is also supported by a JC Bose Fellowship from the Science and Engineering Research Board, Government of India. Apart from financial support, the funding bodies had no role in the design of the study and no role in the collection, analysis, and interpretation of data or in writing the manuscript.

Affiliations

1. CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, 500007, India

   Neha Rajendra Kachewar, Vishal Gupta, Ashish Ranjan, Hitendra Kumar Patel & Ramesh V. Sonti

2. National Institute of Plant Genome Research, New Delhi, 110067, India

   Ramesh V. Sonti

3. Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI, 53706, USA

   Ashish Ranjan

Contributions

NRK, VG and RVS designed the research. NRK, VG and AR performed the research. NRK analyzed the data and wrote the manuscript. NRK, HKP and RVS edited the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence toRamesh V. Sonti.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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