Loose Plant Architecture 1-Interacting Kinesin-like Protein KLP Promotes Rice Resistance to Sheath Blight Disease

doi:10.21203/rs.3.rs-330525/v1

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Loose Plant Architecture 1-Interacting Kinesin-like Protein KLP Promotes Rice Resistance to Sheath Blight Disease

https://doi.org/10.21203/rs.3.rs-330525/v1

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Background

Sheath blight disease (ShB) is a destructive disease affecting rice production. Previously, we have reported that Loose Plant Architecture 1 (LPA1) promotes resistance to ShB. However, the mechanisms by which LPA1 confers resistance against this disease have not been extensively investigated. Notably, interactors that regulate LPA-1 activity remain elusive.

Findings

In this study, we identified the interaction of kinesin-like protein (KLP) with LPA1 in the nucleus of rice cells by yeast two-hybrid, bimolecular fluorescent complimentary (BiFC), and co-immunoprecipitation (co-IP) assays. To investigate the role of KLP in promoting resistance to ShB, wild-type, klp mutant, and KLP overexpressor (KLP OX) rice plants were inoculated with Rhizoctonia solani AG1-IA. The results indicated that, compared with the wild-type control, klp mutants were more susceptible while KLP OX plants were less susceptible to ShB. Since LPA1 transcriptionally activates PIN-FORMED 1a (PIN1a), we examined the expression of 8 related PIN genes. The results showed that only the expression of PIN1a and PIN3b coincided with KLP expression levels. In addition, a chromatin immunoprecipitation (ChIP) assay showed that KLP bound directly to the promoter region of PIN1a but not of PIN3b. Transient expression assays confirmed that LPA1 and KLP transcriptionally activate PIN1a, and that coexpression of KLP and LPA1 had an additive effect on the activation of PIN1a, suggesting that KLP enhances LPA1 transcriptional activation activity.

Conclusions

Taken together, our results show that KLP is a novel LPA1 interactor that promotes resistance of rice to ShB.

Plant Molecular Biology and Genetics

Plant Physiology and Morphology

KLP

sheath blight disease

transcription activation

defense

rice

Rhizoctonia solani (R. solani) is a causative agent of sheath blight disease (ShB) in rice (Oryza sativa) that severely affects rice production in China (Savary 1995). Damage inflicted by ShB occurs during the entire rice cultivating period, and mainly affects the leaves, sheaths, and panicles (Savary 1995). When the disease is severe ShB reduces the yield by up to 50% (Savary et al. 2000). Nowadays, fungicide application is the main approach to control ShB, due to a lack of resistant cultivars and resistance-related genes (Savary et al. 2000). However, the use of pesticides results in severe pollution and increases the cost of cultivation. Therefore, there is an urgent need to identify resistance-related genes and to use those genes to obtain resistant rice cultivars to protect rice from ShB.

Extensive studies have shown that overexpression of chitinase, β-1,3-glucanase, and polygalacturonase inhibiting protein1 (OsPGIP1) could enhance the resistance of rice to R. solani (Shah et al. 2009; Mao et al. 2014; Wang et al. 2015). Inducible expression of OsACS2, an 1-aminocyclopropane-1-carboxylic acid (ACC) synthetase that is a key enzyme in ethylene synthesis, promotes rice resistance to blast and sheath blight (Helliwell et al. 2013). Overexpression of BROAD-SPECTRUM RESISTANCE2 (BSR2) has been shown to increase rice resistance to R. solani (Maeda et al. 2019). Salicylic acid-triggered defense mechanisms play an important role in resistance to R. solani (Kouzai et al. 2018). OsARS2, Os2H16, and OsGSTU5 are positive regulators of resistance of rice to ShB (Tiwari et al. 2020; Li et al. 2018), while OsARS2 directly regulates Os2H16 via binding of a GT1 cis-element in the promoter region (Li et al. 2018). A genome-wide association study identified the F-box protein ZmFBL41 as a negative regulator of the resistance of maize to banded leaf and sheath blight through its interaction with ZmCAD, a monolignol biosynthesis enzyme. The rice homologous gene OsCAD8b plays a similar function in the defense against ShB (Li N 2019). Our recent work demonstrated that the sugar transporter 11 (SWEET11) negatively regulates the defense of rice against ShB (Gao et al. 2018), while the transcription factor DOF11 activates SWEET14 promoting resistance of rice to ShB (Kim et al. 2020). This is related to ABI3/VP1-Like 1 (RAVL1) that positively regulates the defense of rice against ShB by modulation of brassinosteroids and ethylene signaling (Yuan et al. 2018). Overexpression of Loose Plant Architecture 1 (LPA1), containing an indeterminate domain (IDD), promoted the defense of rice against ShB via activation of PIN1a (Sun et al. 2019). Furthermore, IDD13, IDD3, and the G-protein γ subunit DEP1 interact with LPA1 to differentially regulate the resistance of rice to ShB (Miao Liu et al. 2020; Sun et al. 2020). However, the mechanism by which LPA1 regulates resistance against ShB remains to be investigated.

To investigate the mechanism by which LPA1 regulates the resistance of rice to ShB, we performed a yeast two-hybrid (Y2H) screen. Among potential LPA1 interactors, we identified a kinesin-like protein (KLP). The Y2H results indicated that LPA1 interacts with KLP and IDD13 (Fig. 1a). Furthermore, a split-GFP assay was performed in rice protoplasts, confirming that LPA1 interacts with KLP in the nucleus, while no visible signal was detected in the negative control (LPA1-nYFP+cYFP) (Fig. 1b). In addition, a co-IP was carried out where KLP-Myc was coexpressed with LPA1-GFP in N. benthamiana leaves, and an anti-GFP antibody was used to immunoprecipitate LPA1-GFP. Western blot analysis using an anti-Myc or anti-GFP antibody indicated that KLP-Myc and LPA1-GFP were successfully expressed and that LPA1 also interacts with KLP in plants (Fig. 1c). Since LPA1 expression was induced by inoculation of R. solani, we also examined KLP expression upon inoculation with R. solani. qRT-PCR data showed that LPA1 was induced after 72 hours of the inoculation, but R. solani inoculation did not change the expression levels of KLP (Fig. 1d).

To analyze the role of KLP in promoting resistance of rice to ShB, klp mutants and KLP overexpression lines were generated. Two independent klp mutants named klp-1 and klp-2, were generated by insertion of T-DNAs into the 11^th intron (Fig. 2a). qRT-PCR data showed that no KLP transcripts were detected in klp-1 and klp-2 mutant plants (Fig. 2b). In parallel, the KLP expression level was examined in wild-type and 4 KLP overexpressors (KLP OX) lines (#1, #2, #3, and #5). The qRT-PCR data showed that KLP expression levels were higher in KLP1 OXs compared with wild-type plants, and the highest expression was detected in KLP OX #5 (Fig. 2c). Inoculation with R. solani AG1-IA revealed that, compared with wild-type plants, klp mutants (klp-1 and klp-2) were more susceptible (p<0.05) while KLP OX plants (#2 and #5) were less susceptible (p<0.05) to ShB (Fig. 2d). The percentage of the leaf area covered with lesions was 39.1% in WT, 48.2% in klp-1, 47.2% in klp-2, 27.5% in KLP OX #2, and 26.5% in KLP OX #5 plants (Fig. 2e).

Previously, we have found that LPA1 regulates the resistance of rice to ShB by directly activating PIN1a expression. To test whether KLP also regulates PIN gene expression, the expression levels of 8 PIN genes were analyzed in wild-type, klp-1, and KLP OX-5 plants. The results showed that PIN1a and PIN3b expression levels were suppressed in klp-1 and had increased in KLP OX-5 plants compared to wild-type plants. PIN1b, PIN1c, and PIN3a expression levels were suppressed in both klp-1 and KLP OX-5 plants compared to wild-type plants. PIN5a and PIN5b expression levels were higher in KLP OX-5 compared to wild-type plants, while no differences in PIN5a and PIN5b expression levels were observed between wild-type and klp-1 plants. Meanwhile, the expression level of PIN1d was similar between wild-type, klp-1, and KLP OX-5 plants (Fig. 3).

Since PIN1a and PIN3b expression was positively regulated by KLP, the affinity of KLP to PIN1a and PIN3b promoters was examined. Three regions within the 1.5 kb promoter regions of PIN1a (P1-P3) and PIN3b (P4-P6), respectively (Fig. 4a), were tested by ChIP PCR using KLP-GFP transgenic plants. The immunoprecipitation was performed using the pre-immune (control) and anti-GFP antiserum. The ChIP-PCR results showed that KLP directly bound to the P3 region of the PIN1a promoter, but no binding affinity was observed in PIN3b promoter region (Fig. 4b). To verify that LPA1 and KLP bind to the P3 region of the PIN1a promoter and activate its expression, transient expression assays were performed using rice protoplasts. The 35S:LPA1, 35S:KLP, or 35S:LPA1+35S:KLP plasmids were cotransformed with a construct expressing the ß-glucuronidase gene (GUS) under the control of the 1.5 kb pPIN1a promoter in the protoplasts. A 35S:Luc (luciferase) plasmid was used as the internal control for evaluation of transformation efficiency (Fig. 4c). Transient assay results showed that LPA1 and KLP activated pPIN1a, and that LPA1 had a higher pPIN1a activation activity than KLP. Interestingly, coexpression of LPA1 and KLP resulted in a stronger transcriptional activation of pPIN1a than expression of either LPA1 or KLP alone (Fig. 4d), indicating an additive effect of KLP on LPA1-mediated activation of pPIN1a.

The isolation of resistance-related genes and the breeding of rice plants using these genes is the most efficient way to control disease-mediated loss in rice production. ShB is a destructive rice disease that causes severe yield reduction. However, the molecular mechanism remains to be determined. Previously, we reported that the IDD-containing protein LPA1 promotes resistance to ShB. In the current study, we have shown that KLP interacts with LPA1 in the nucleus, which was verified by yeast two-hybrid, split-GFP, and co-IP assays. Further genetic analysis using inoculation of KLP mutants and overexpressing plants with of R. solani AG1-IA strain revealed that KLP promotes rice resistance to ShB. Two independent alleles of klp mutants were more susceptible while two KLP OXs were more resistant to ShB compared to wild-type plants. These results suggest that KLP is an LPA1-interacting protein that positively regulates the defense of rice against ShB. Furthermore, our qPCR results demonstrated that PIN1a and PIN3b expression levels positively correlated with KLP levels, while the expression of other PIN genes was differentially regulated by KLP. A ChIP assay using KLP-GFP transgenic plants revealed that KLP directly bound to the PIN1a but not to the PIN3b promoter region. It has been previously reported that the kinesin-like protein BRITTLE CULM12 (BC12) directly binds to the KO2 promoter of the gibberellic acid (GA) biosynthesis gene directly regulating its expression (Li et al. 2011), indicating that a KLP-type protein can function as a transcriptional regulator. Further transient assays confirmed that KLP and LPA1 activate a 1.5 kb fragment containing the PIN1a promoter, and KLP plays an additive function in LPA1-mediated PIN1a activation. KLP is not transcriptionally activated by infection of R. solani, implying that KLP-mediated rice resistance to ShB might be through activation of downstream gene expressions. PIN1a is a polar auxin transporter, and genetic studies have revealed that PIN1a positively regulates the defense mechanism against ShB in rice. In addition, exogenous treatment of auxin promotes the resistance of rice to ShB (Sun et al. 2019), suggesting that KLP might regulate PIN1a transcription to modulate local auxin content resulting in increased resistance.

In conclusion, we have shown that KLP, a keratin-like protein, interacts with transcription factor LPA1 to activate downstream gene expression in a dosage-dependent manner. Our analyses demonstrated that KLP and LPA1 together directly activate PIN1a expression. PIN1a controls auxin transport to modulate auxin distribution, and the increase of local auxin concentration promotes resistance of rice to ShB. Taken together, our results suggest that KLP partners with LPA1, to promote resistance rice to ShB via activation of PINa-dependent auxin redistribution and subsequent activation of auxin signaling.

KLP: Keratin-like protein; IDD: Indeterminate domain; ShB: Sheath blight disease; PGIP1: Polygalacturonase inhibiting protein; PIN: PIN-FORMED; LPA1: Loose plant architecture1; SWEET11: Sugar will be eventually exported transporter; RAVL1: Related to ABI3/VP1-Like 1; WT: wild type; Overexpressor: OX; ChIP: Chromatin-immunoprecipitation; Co-IP: Co-immunoprecipitation; BC12: BRITTLE CULM12; GA: gibberellic acid; GUS: ß-glucuronidase; Luc: Luciferase; Y2H: Yeast two-hybrid

Acknowledgments

The authors would thank Dr. Chang-deok Han at Gyeongsang National University, Korea for providing rice seeds.

Authors’ contributions

J Chu and YH Xuan designed the experiment and wrote the manuscript. J Chu conducted the experiments and performed data analysis. J Chu and YH Xuan corrected the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the Nature Science Foundation of Liaoning (2020-YQ-05), Nature Science Foundation of China (32072406), and National Key R&D Program of China (2018YFD02002).

Availability of data and materials

The datasets supporting the conclusions of this article are provided within the article and its additional files.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no conflict of interest.

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Loose Plant Architecture 1-Interacting Kinesin-like Protein KLP Promotes Rice Resistance to Sheath Blight Disease

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