Minfeng Lu, Jinhui Chen, Hn Meng, Gungling Mo, Yunhong Liu, Fengping Chen,Zonghu Wng, Mo Wng,d,*

a Fujian Universities Key Laboratory for Plant-Microbe Interaction, Ministerial and Provincial Joint Innovation Centre for Safety Production of Cross-Strait Crops, College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China

b Fujian Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China

c Fuzhou Institute of Oceanography, Minjiang University, Fuzhou 350108, Fujian, China

d State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming 650201, Yunnan, China -

Keywords:Ferredoxin Rice OsFd4 Oxidative stress Blight bacteria

ABSTRACT Ferredoxins (Fds) in plastids are the most upstream stromal electron receptors shuttling electrons to downstream metabolic systems and function in various physiological processes of dicots, but their roles in monocots’ response to stresses are still unclear.In this study, the functions of OsFd4, the major non-photosynthetic type Fd in rice, were characterized under oxidative stress and Xanthomonas oryzae pv.oryzae(Xoo)infection.OsFd4-knockout mutants displayed no defects in key agronomic traits and blast resistance,but were more sensitive to hydrogen peroxide(H2O2)treatment than the wild type.Transient expression of OsFd4 alleviated H2O2-induced rice cell death, suggesting that OsFd4 contributes to rice tolerance to exogenous oxidative stress.Deletion of OsFd4 enhanced rice immune responses against Xoo.OsFd4 formed a complex in vivo with itself and OsFd1, the major photosynthetic Fd in rice, and OsFd1 transcripts were increased in leaf and root tissues of the OsFd4-knockout mutants.These results indicate that OsFd4 is involved in regulating rice defense against stresses and interplays with OsFd1.

1.Introduction

Because plants are challenged by various biotic and abiotic stresses throughout their growth,they must perceive and respond to unfavorable conditions and pathogen attack at the cellular level to avoid severe physiological disorders or death [1].This process involves complex and elaborate signaling pathways,including production of reactive oxygen species(ROS),ion influx and efflux,activation of various kinase cascades, and accumulation of stress hormones [2,3].ROS, comprising mainly superoxide radical (O2-)and hydrogen peroxide (H2O2), are rapidly generated in plants as they sense stresses and serve as cellular signaling molecules for activating defense mechanisms [4].Persistent or intense environmental challenges cause plants to suffer oxidative stress because excessive ROS accumulation irreparably damages cellular molecules.

Besides their roles in photosynthesis and synthesis of metabolites,plastids act in mediating plant responses to abiotic and biotic stresses, by hosting biosynthesis of key defensive molecules including ROS, salicylic acid (SA), and jasmonic acid (JA), and regulating cellular redox homeostasis [5–8].Ferredoxins (Fds) are a group of small iron-sulfur [2Fe-2S] cluster-containing proteins localized in plastid stroma, which function as the most upstream electron carriers distributing electrons to various acceptors for downstream metabolic reactions [9].Based on their expression pattern, Fds in higher plants are classified into photosynthetic and non-photosynthetic types.The former receives electrons from photosystem I and the later uses NADPH generated in the oxidative pentose phosphate pathway as an electron donor and usually has a higher redox potential [9,10].When the major Fds in plastids are defective, the electron transfer flows become blocked, causing transfer of excess electrons to O2or H2O, forming ROS [11].

Fds are involved in regulating plant responses to various stresses.In Arabidopsis, deletion of AtFd2,which contributes about 90%of total leaf Fd,led to ROS accumulation in chloroplasts[12].AtFd2-knockout mutant (AtFd2-KO) plants adapted better to long-term high-light conditions by induction of genes conferring photoprotection [13].Overexpression of the endogenous Fdencoding gene in Chlamydomonas reinhardtii reduced endogenous H2O2production and increased tolerance to heat stress [14].Similar results were found[15]when the leaf-type ferredoxin of sweet pepper was expressed in Arabidopsis.Fds are also engaged in regulating plant immunity.Ectopic expression of sweet pepper ferredoxin in Arabidopsis [16] or tobacco [17] increased resistance of the transgenic plants to compatible pathogens, and the AtFd2-KO mutant displayed more susceptibility to (hemi)biotrophic pathogens due to the higher accumulation of JA and its derivatives after infection compromising SA signaling pathway-mediated resistance[11].

Rice is a worldwide staple cereal crop and also serves as a model monocot species for functional genomics studies.Bacterial blight caused by Xanthomonas oryzae pv.oryzae (Xoo) is the most devastating bacterial rice disease worldwide,causing yield losses as high as 50%[18].In rice,five Fds,OsFd1–OsFd5,have been identified,of which OsFd1 is the major leaf-type Fd and functions in ensuring rice survival at seedling stage [19].OsFd4 is regarded as the main non-photosynthetic type of Fd in rice, as its transcript levels are highest in non-photosynthetic tissues [10], whereas the biological functions of OsFd4 are still ambiguous.In this study, via creating OsFd4-knockout mutants and biochemical investigation, we determined the important role of OsFd4 in rice responses to oxidative stress, and found that OsFd4 functions as a negative regulator in rice resistance against Xoo.Moreover, the association between OsFd4 and OsFd1 was identified.

2.Materials and methods

2.1.Plant materials and growth conditions

Knockout of OsFd4(LOC_Os03g61960)in the rice japonica cultivar Zhonghua 11 (ZH11) was performed by CRISPR/Cas9 geneediting technology as described [20].The selected guide RNA(gRNA) target sequence of OsFd4 was 5′-gggcaagagcacgagttcgagg-3′.Germinated rice seeds were grown in a chamber at 28 °C with a 12-h photoperiod (600–800 μmol m-2s-1) and 70% relative humidity.Rice plants grown in the paddy at Fuzhou were used for investigating the agronomic traits.

2.2.H2O2 treatment

Rice seeds were germinated and grown on 1/2 MS medium for 12 days.The seedlings were cultured in a 0.5× Kimura B nutrient solution [21] with 0.02% H2O2or with water as a mock treatment for 10 days in a growth chamber.To measure the levels of rice cell death induced by H2O2treatment, a ProUBQ:LUC reporter was cotransfected with OsFd4-GFP plasmids or empty pYBA1132-GFP vector (as a control) into rice protoplasts.After 16 h, 0.02% H2O2or water (as a mock control) was added into the protoplast suspension.One hour later, LUC activity was measured after addition of D-luciferin using a GloMax Navigator Microplate Luminometer(Promega, USA).

2.3.Xoo and Magnaporthe oryzae inoculation

Field-grown rice plants at tilling stage were inoculated with Xoo strain PXO86 using the leaf-clipping method[22],and blight lesion length and bacterial population in the infected leaves were investigated following Tian et al.[23].To measure transcript levels of defense-responsive genes post-Xoo infection, four-week-old rice plants grown in a greenhouse were inoculated with PXO86, and inoculum without blight bacteria was used as a mock control.

Spray inoculation of 3-week-old rice seedlings with M.oryzae strain Guy11 was performed as described previously [24].At five days post-inoculation (dpi), the diseased leaves from three plants were imaged and used for estimating the proportion of lesion area with ImageJ 1.51j8 software (National Institutes of Health,Bethesda, MD, USA).

2.4.ROS burst detection and callose deposition observation

The method of measuring the flagellin epitope flg22-induced ROS burst was as described [23] with minor modifications.Leaf sheaths(～3 mm in length)from 10-day-old rice seedlings growing on 1/2 MS medium were collected and floated on sterile water overnight for removing the ROS induced by cutting.They were then incubated with 100 μL reaction solution (20 μmol L-1luminol, Sigma-Aldrich, USA, Cat.No.123072 and 2.5 μg mL-1peroxidase, Solarbio, China, Cat.No.P8020) containing 100 nmol L-1flg22 for detecting the ROS burst.Luminescence was measured with a Mithras luminometer (Berthold, Germany) every minute for 1 h.Five replicates were performed for each sample.To observe callose deposition, the leaves of seven-day-old rice seedlings were detached and treated with 100 nmol L-1flg22.The assay was performed as previously described [25].Callose deposition on the leaves was observed under UV light (340–380 nm; Zeiss LSM880,Germany), and the callose dots in the resulting images were counted with ImageJ software.

2.5.Gene expression measurement

Total RNA was extracted from rice leaves using Trizol reagent(Ambion,USA,Cat.No.15596018)according to the manufacturer’s instructions.cDNA was synthesized with the Evo M-MLV RT kit(Accurate Biology, China, Cat.No.AG11711).qRT-PCR assays were performed using MonAmp ChemoHS qPCR Mix(Monad,China,Cat.No.MQ00401S),with a CFX96 Touch Real-Time PCR Detection System(Bio-Rad,USA).The 2-ΔΔCTmethod[26]was used for calculating relative expression with OsUBQ as internal control.All primers used for qRT-PCR analysis are listed in Table S2.

2.6.Protein interaction and subcellular localization assays

A yeast two-hybrid (Y2H) system (Clontech, USA, Cat.No.630489)was used for investigating the interaction between OsFds.The coding regions of OsFd1 and OsFd4 were cloned into pGBKT7(BD) or pGADT7 (AD).The construct combinations were transformed into yeast strain AH109 following the manufacturer’s instructions, and then the yeast clones were transferred onto SD/-Trp-Leu-His-Ade medium to detect interaction.To perform split-LUC complementation and co-IP assays,the indicated combinations of constructs were transiently co-expressed in Nicotiana benthamiana(N.benthamiana)leaves by Agrobacterium tumefaciens strain GV3101-mediated infiltration.The split-LUC complementation and co-IP assays were performed as previously described[24].Immunoblotting was performed with anti-GFP (TransGen,China, Cat.No.HT801) and anti-HA (Abmart, China, Cat.No.M20003S) antibodies and anti-mouse secondary antibody (Abbkine,China,Cat.No.A21010)diluted as the manufacturer’s instructions.To identify the subcellular location of OsFd4, the coding region of OsFd4 was cloned into the pYBA1132-GFP vector (with a GFP tag at the C terminus), and the subcellular localization of OsFd4-GFP was investigated by transient expression in rice protoplasts and N.benthamiana leaves.Free GFP was expressed as a control.Fluorescence signals were observed with a confocal microscope(Zeiss LSM880,Germany).All primers used for plasmid construction are listed in Table S1.

2.7.Gene accession numbers and primer sequences

Sequence of the genes in this article can be found in the Rice Genome Annotation Project database (https://rice.uga.edu/) under the following accession numbers:OsFd1(LOC_Os08g01380),OsFd4(LOC_Os03g61960), AOS2 (LOC_Os03g12500) [27], OsPR5(LOC_Os12g43380) [28], OsCht1 (LOC_Os06g51060) [29], OsPAL4(LOC_Os02g41680) [30], OsUBQ (LOC_Os03g13170).

3.Results

3.1.Knockout of OsFd4 did not affect rice agronomic traits

To characterize the expression pattern of OsFd4 in the ZH11 plants, tissues of ZH11 were harvested at booting stage.As shown in Fig.S1, the transcript level of OsFd4 in roots was much higher than that of OsFd1, confirming that OsFd4 is the major nonphotosynthetic Fd in ZH11.OsFd4 transcripts were also detected in leaf and young panicle tissues, where OsFd1 transcripts were predominant (Fig.S1).Although OsFd4 was not clustered with the leaf-type Fd group in a phylogenetic tree [19], it displays high similarity to OsFd1 and AtFd2 (Fig.S2).When transiently expressed in ZH11 protoplasts (Fig.S3A) and N.benthamiana(Fig.S3B), OsFd4 localized to chloroplasts.These findings suggest a role for OsFd4 in distributing electrons generated from photosynthesis in leaf tissues.To identify its functions, we knocked out OsFd4 in ZH11 by CRISPR/Cas9-mediated gene editing.Among the transgenic plants of T0 generation, three homozygous allelic OsFd4-knockout mutants, named Osfd4-1, Osfd4-2 and Osfd4-3,were obtained (Fig.1A).When grown in the field, these Osfd4 mutants of T1 generation displayed no obvious defects in growth in comparison with the wild type (Fig.S4).They were also similar to ZH11 in panicle morphology and the yield traits including seedsetting rate, grain size and weight (Fig.S5), suggesting that OsFd4 plays dispensable roles in rice growth and development under normal conditions.

3.2.Osfd4 mutants displayed reduced tolerance to exogenous H2O2 treatment

Considering the essential role of Fds in controlling cellular redox status, we wondered whether deletion of OsFd4 leads to defects in rice tolerance to exogenous oxidative stress.To test this,seven-day-old seedlings of ZH11 and Osfd4 mutants were treated with 0.02% H2O2(using water as a mock control) for 10 days.In the mock group,there was no visible difference in growth between wild type and the Osfd4 mutants (Fig.1B).Application of H2O2led to growth inhibition in ZH11 and Osfd4 plants.However,the Osfd4 mutants displayed more sensitivity to H2O2treatment than the wild type (Fig.1B), as both shoot and root length of the mutants were significantly shorter than those of ZH11(Fig.1C,D).The number of lateral roots of the Osfd4 mutant was significantly lower than that of the wild type in response to H2O2treatment (Figs.1E, S6).To further investigate whether OsFd4 expression promotes rice cell tolerance to H2O2application, we co-transfected a construct expressing the firefly luciferase (LUC) reporter gene under the control of maize UBQ promoter (ProUBQ:LUC) together with the OsFd4-GFP or empty GFP(as a control)plasmids into rice protoplasts.Sixteen hours later,0.02%H2O2(using water as a mock control)was applied to the rice protoplasts for 1 h and then LUC activity was measured.H2O2treatment caused a large decrease in the chemiluminescence signals generated by cellular LUC activity,whereas signals from protoplasts expressing OsFd4-GFP were significantly higher than those from protoplasts expressing GFP(Fig.1F, G), suggesting that OsFd4 increased rice cell survival in response to H2O2treatment.Collectively, our results indicate that OsFd4 contributes to rice tolerance to exogenous oxidative stress.

3.3.Deletion of OsFd4 increases rice resistance to Xoo

To investigate whether OsFd4 regulates rice resistance to biotic stresses, we inoculated the wild type and Osfd4 allelic mutants at tillering stage with the Xoo strain PXO86.The Osfd4 mutants displayed significantly shorter blight lesions than ZH11 at 14 days post-inoculation (dpi) (Fig.2A, B).The Osfd4 mutants also supported significantly less Xoo growth than the wild type (Fig.2C).To further investigate the mechanism of increased resistance to Xoo by OsFd4 deletion, the flg22-induced PTI (pathogenassociated molecular pattern-triggered immunity) responses of Osfd4-1 were examined.Leaf sheaths of seven-day-old ZH11 and Osfd4-1 seedlings were treated with flg22, using water as a mock control, and then the ROS burst was measured.Flg22 application elicited ROS bursts in both ZH11 and Osfd4-1 leaves, but Osfd4-1 showed higher levels of ROS burst than ZH11 at the measured time points (Fig.2D).Moreover, significantly more callose was deposited on Osfd4-1 leaves than on ZH11 leaves following flg22 treatment (Fig.2E).Transcript levels of four defense-responsive genes,AOS2 (allene oxide synthase 2), OsPAL4 (phenylalanine ammonialyase 4), OsPR5 (pathogenesis-related 5), and OsCht1 (chitinase 1)were significantly higher in Osfd4-1 than in ZH11 at 2 dpi(Fig.S7).These results indicate that deletion of OsFd4 enhanced rice defense against blight bacteria.After spray inoculation with the rice blast fungal isolate Guy11 conidial spores, we found little difference in disease symptoms between ZH11 and the Osfd4 mutants (Fig.S8), suggesting the dispensable role of OsFd4 in rice resistance to blast fungi.

3.4.The interplay between OsFd4 and OsFd1

As Fds in other species form functional dimers, association among the OsFds was investigated.The Y2H assay indicated a strong interaction between OsFd4 and OsFd1 (Fig.2F).Moreover,OsFd4 can bind with itself (Fig.2F).The co-IP and split-LUC complementation assays further showed that OsFd4 can form a complex with itself and OsFd1 in vivo (Figs.2G, S9).In addition, we found that OsFd1 transcripts were up-regulated in both leaf and root tissues of the Osfd4 mutants,compared with those in the wild type (Fig.S10).Thus, OsFd4 directly interacts with OsFd1, and knockout of OsFd4 resulted in up-regulation of OsFd1 transcription.

Relative to the mock control, OsFd4 transcript levels were initially down-regulated after H2O2application, but slightly and markedly increased at 3 and 7 days post-treatment, respectively(Fig.S11A, left), suggesting that rice plants increase OsFd4 expression to increase their tolerance after long exposure to exogenous oxidative stress.When ZH11 plants at tillering stage were challenged with Xoo, OsFd4 transcripts were significantly increased compared with mock treatment beginning at 2 dpi(Fig.S11B,left).In contrast, OsFd1 transcript levels were decreased by H2O2treatment or Xoo inoculation beginning at 2 days after challenging(Fig.S11A, B, right).

4.Discussion

In rice photosynthetic tissues,transcript levels of OsFd1 are the highest [19], and OsFd4 transcripts are most abundant among the rest OsFds [10], suggesting that OsFd4 is the secondary major leaf-type Fd in rice.Although the plastids in non-photosynthetic cells lack the capacity for photoreduction, the Fds can obtain electrons from NADPH via Fd-NADP+oxidoreductase,which is reversed electron transfer in photosynthetic tissues [31].As the major type of non-photosynthetic Fd, OsFd4 was expected to function in distributing electrons to the enzymes of bioassimilatory and biosynthetic processes in non-photosynthetic tissues.Thus, deletion of OsFd4 should block electron transfer flows in photosynthetic and non-photosynthetic tissues, causing transfer of excess electrons to O2or H2O to form ROS [32].This defect should be more severe in rice root cells,where OsFd4 is the major form of Fd.In this study,we found that deletion of OsFd4 compromised rice plants tolerance to exogenous oxidative stress, which is largely due to the higher basal ROS accumulation in root cells of OsFd4-knockout mutants.

ROS accumulation is a double-edged sword in plant cells,causing cellular damage and toxic effects at high concentration but acting as a signaling molecule to enhance resistance at low concentration [33].Consistently, Osfd4 mutants displayed increased resistance to blight bacteria(a biotroph),probably owing to the increased ROS accumulation in their leaves.The finding that knockout of OsFd4 resulted in no penalties to rice agronomic traits and yield suggests the potential of utilizing OsFd4 gene editing in rice resistance breeding against bacterial blight disease.After Xoo inoculation,transcripts of OsFd4 were increased in rice leaves.Considering the role of OsFd4 in rice immune responses against blight bacteria, we hypothesize that some effector(s) secreted by Xoo functions to disrupt rice immunity by inducing OsFd4 expression.However, knockout of OsFd4 did not contribute to resistance against the blast fungal isolate Guy11.Given that the blast fungus is a hemibiotrophic pathogen, the mild ROS increase in Osfd4 leaves may have little effect on inhibiting blast fungal extension.

Pyrococcus furiosus Fd forms a functional dimer, and its monomer is impaired in electron transfer, implying that the complex form can increase the efficiency of Fd in electron carrying and delivering [34].Similarly, the Fd in Pseudomonas putida was later found to form a tail-to-tail dimer [35].The interaction, in our study,of OsFd4 with itself and OsFd1 suggests that the mechanism of association among Fds to facilitate electron transfer is conserved in the plant kingdom.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig.2.OsFd4 functions as a negative regulator in rice resistance against Xoo and associates with itself and OsFd1.(A)ZH11 and Osfd4 plants were inoculated with Xoo strain PXO86 and diseased leaves were photographed at 14 dpi.Scale bar, 3 cm.(B) The lesion length of ZH11 and Osfd4 mutants, with bars representing means ± SD (n = 6).(C)Blight bacterial populations grown in diseased leaves of ZH11 and Osfd4 mutants.Bars represent means ± SD (n = 3).These assays were performed in three independent replicates with similar results.(D) Leaf discs of ZH11 and Osfd4-1 seedlings were treated with 100 nmol L-1 flg22, after which the resulting ROS burst was detected at the indicated time points.Error bars represent SD(n=5).(E)Callose deposition on ZH11 and Osfd4-1 leaves after flg22 treatment was imaged with a microscope under UV light(left).Scale bars, 30 μm.Mean number of callose deposits per view was calculated with ImageJ (right) with bars representing means ± SD(n =5).Results in (D)and (E) are from one of three independent experiments with similar results.*,P<0.05;**,P<0.01(Student’s t-test);ZH11,Zhonghua 11.(F)The Y2H assays showing that OsFd4 interacts with itself and OsFd1.The combinations with the empty vectors BD(pGBKT7)or AD(pGADT7)were used as negative controls.(G)In vivo association of OsFd4 with itself and OsFd1 was determined by co-IP assays via Agrobacterium-mediated transient expression in N.benthamiana leaves.The combination of GFP and OsFd4-HA co-expression served as a negative control.IP was performed with anti-GFP beads, and immunoblotting was conducted with anti-GFP and anti-HA antibodies.

CRediT authorship contribution statement

Minfeng Lu:Investigation,Formal analysis,Visualization,Writing – original draft.Jinhui Chen:Investigation, Formal analysis,Visualization, Writing – original draft.Han Meng:Investigation.Guangling Mo:Investigation.Yunhong Liu:Investigation.Fengping Chen:Writing – review & editing.Zonghua Wang:Writing– review & editing.Mo Wang:Conceptualization, Methodology,Validation, Funding acquisition, Project administration, Resources,Supervision, Writing – original draft.

Acknowledgments

This research was supported by the National Natural Science Foundation of China (31701777), the National Natural Science Foundation for International Exchanges (NSFC-RS)(31911530181), the Fujian Provincial Science and Technology Key Project (2022NZ030014), and Key Plant Protection Disciplinary Development Project (Fujian Agriculture and Forestry University,103-722022001) to Mo Wang.

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2023.04.003.