Zhongchi Cui,Fng Ling,Jirui Zhng,Fei Wng,Dqun Liu,b,Hiyn Wng,*

a Technological Innovation Center for Biological Control of Crop Diseases and Insect Pests of Hebei Province,College of Plant Protection,Hebei Agricultural University,Baoding 071000,Hebei,China

b Graduate School of Chinese Academy of Agricultural Sciences,Beijing 100081,China

Keywords:Bipolaris sorokiniana Disease resistance Puccinia triticina Transgenic wheat

ABSTRACT Thaumatin-like protein (TLP) plays an important role in combating plant pathogen infection.Common root rot caused by Bipolaris sorokiniana and leaf rust caused by Puccinia triticina(Pt)are major fungal diseases in wheat.The disease responses of TaTLP1-overexpressing transgenic lines (TaTLP1-OE) were evaluated after inoculation with each pathogen.The TaTLP1-OE lines had no apparent differences in tiller number and 1000-kernel weight from the wild type Jinan Wheat No.1(JW1),whereas resistance to leaf rust and common root rot was improved,resulting from activated peroxidase and β-1,3-glucanase after B.sorokiniana infection,and reactive oxygen species-related genes were upregulated in TaTLP1-OE lines after Pt infection.These results indicated that stable expression of TaTLP1 increased resistance against both diseases.

1.Introduction

Plants respond to external pathogen attack by activating innate immune responses.Pathogenesis-related (PR) proteins play an essential role in the plant defense system as indispensable components of the innate immune response [1].Thaumatin-like protein(TLP) belongs to a PR family that shares high sequence homology to thaumatin,a sweet-tasting protein inThaumatococcus danielli[2].TLP confers antifungal activity in plants by inhibiting spore germination and mycelial growth[3].TLP-overexpression in grapes and rice confers host resistance to fungal pathogens [4,5].However,stable transgenic expression ofTLPin wheat against fungal pathogens remains to be established.

Wheat is a leading food crop with a large and complex genome.However,fungal diseases including common root rot (CRR)caused byBipolaris sorokinianaand leaf rust caused byPunccinia triticina(Pt) are significant constraints in major wheatproducing regions worldwide.CRR causes significant yield losses ranging from 15% to 20% in individual fields [6].However,a lack of germplasm providing resistance to CRR is a challenge for breeding resistant wheat varieties.Leaf rust remains one of the most destructive diseases of wheat and can cause 20%–40% yield losses under favorable conditions [7].Wheat defense-related genes such as PR genes,that participate in leaf rust response have been widely studied [8].However,the molecular mechanisms underlying the action of PRs remain largely unexplained.TaTLP1(GenBank accession number KJ764822)was originally cloned from TcLr19 in response to infection byPtrace PHNT [9].There are three gene homologs ofTaTLP1in the wheat genome,located on chromosomes 7A,7B and 7D.A recent study found that interaction of TaTLP1 and TaPR1 contributed to wheat defense responses to leaf rust and that cosilencing ofTaTLP1andTaPR1significantly reduced resistance to leaf rust compared to single silencing of either gene [9].These experiments demonstrated that geneTaTLP1is tightly associated with rust resistance in wheat.TaTLP1was transformed into vector pLGY-02 under the control of the maize ubiquitin promoter (ubi) and transferred into cultivar Jinan Wheat No.1 (JW1) by the Agrobacterium-mediated method[10].In this study,the transgenic wheat lines overexpressingTaTLP1were used to assess the response ofTaTLP1transgenic lines to leaf rust and CRR,and the molecular mechanisms underlying resistance induced by overexpression ofTaTLP1to leaf rust and CRR were investigated,providing new insights into understanding the role ofTaTLP1in defense against fungal pathogens.

2.Materials and methods

2.1.RNA extraction and real-time quantitative PCR (qPCR)

Total RNA was extracted using the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa).Expression ofTaTLP1was investigated using the wheat glyceraldehyde-3-phosphate dehydrogenase (GAPDH,GenBank accession No.AF251217) gene to calibrate its expression level as previously described [11].The 2-ΔΔCT method was employed to quantify relative gene expression levels [12].Statistical significance of differences was calculated using Student’st-test in SPSS 21.0.Data were expressed as means ± SE of three independent experiments.

2.2.Fungal inoculation and histological observation of fungal growth

Six independent T4lines of transgenic wheat was used for segregation analysis.Seedlings (14 days old) ofTaTLP1-OE lines and JW1 were inoculated withPtaccording to methods described previously [9] and disease responses were recorded according to Roelfs[13].Leaf rust severity was measured by number of infection sites in a region of 5 cm2in inoculated leaves and three inoculated leaves were randomly selected per line.Conidiospores ofB.sorokinianacollected from PDA medium were sprayed onto leaves ofTaTLP1-OE and JW1 at the three-leaf stage.Infection type (IT)and disease index (DI) of wheat root rot were scored according to the previous methods[14].Samples were stained by the Rohringer fluorescent staining method to identify fungal tissues and dead cells in the leaf mesophyll of infected plants [15].Stained tissues were observed by fluorescence microscopy.All assays were performed in triplicate.

2.3.Detection of reactive oxygen species and disease resistance-related enzymes activity

Reactive oxygen species (ROS) produced after inoculation withPtquantified by the 3,3′-diaminobenzidine (DAB)staining method to detect the accumulation of H2O2[16].Activities of the ROSrelated enzymes catalase (CAT) and superoxide dismutase (SOD)were measured according to previously published methods [17].Transcripts of ROS producing enzyme-related genes(TaSOD,TaCATandTaNOX)were measured by qPCR.Three seedlings per biological replicate were used for these measurements.Disease resistancerelated enzymes β-1,3-glucanase and peroxidase(POD)were measured after inoculation withB.sorokiniana.β-1,3-glucanase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method with some modifications [18].Reaction mixtures containing laminarin were used as the substrate in β-1,3-glucanase assays;reducing sugars released in the test reaction mixtures were measured with a spectrophotometer at 550 nm.POD activity was determined following Bisswanger et al.[19].Data were expressed as means of triplicate tests of three independent biological samples.

3.Results

3.1.Generation of homozygous transgenic wheat stably expressing TaTLP1

Six transgenic positive T0wheat lines in JW1 background were generated under control of the Ubi promoter (Fig.S1A).Southern blotting with a digoxin-labeledTaTLP1fragment used as a DNA probe binding toTaTLP1to detect insertion copies revealed that 1 or 2 hybridization signals were present in the genomic DNA of transgenic lines,and no hybridization signal was detected in the JW1 wheat genome (Fig.S1B).After segregation analysis based on PCR with primers pLGY-F3 and pLGY-R2 (Table S1),homozygous T4lines of transgenic wheat were retained for further analysis(Table S2).PCR results showed a band with expected size in all six transgenic lines (Fig.S1C),indicating that theTaTLP1gene had been inserted into the wheat genome.Quantification ofTaTLP1expression using qPCR (Fig.1A) showed that expression ofTaTLP1in lines 4,5 and 6 was significantly higher than in JW1.These results indicated thatTaTLP1was stably expressed in the transgenic lines.

To determine whether overexpression ofTaTLP1affected general plant phenotype,the spike lengths and 1000-kernel weight of 30TaTLP1-OE T4plants from 6 T0lines were measured,and there was no significant difference between the WT andTaTLP1-OE lines(Table S3).Physiological development ofTaTLP1-OE lines was similar to JW1 during the entire growth cycle(Fig.1B).Based on these results,we concluded that there were no phenotypic differences betweenTaTLP1-OE lines and JW1 plants.

3.2.TaTLP1-OE lines exhibit resistance to Pt

To assess whetherTaTLP1overexpression altered the response to leaf rust,TaTLP1-OE lines 4,5,6 and the JW1 control were inoculated withPtisolate 07-10-426-1(pathotype PHNT).As shown in Fig.2A,theTaTLP1-OE lines showed a high level of resistance compared to JW1 at two weeks post inoculation(Table S4).Pronounced DAB staining provided evidence of strong H2O2activity in the infected leaves ofTaTLP1-OE lines.By contrast,the infected leaves of WT plants showed limited and weak staining (Figs.2B and S2).Confocal microscopy using a fluorescent brightener staining to visualize fungal development showed that a hypersensitive response (HR) and no visible germination tubes in inoculated leaves ofTaTLP1-OE compared to the fungal development in JW1 leaves at 24 hpi (hours post inoculation) (Fig.2C,D).At 36 hpi,mycelia were restricted to the first few mesophyll cells with few haustoria being formed.At 48 hpi,many secondary hyphal growths with the increasing formation of haustoria were observed in JW1 leaves,but only sparse sporulation,less mycelial growth and mycelial branching,and lower numbers of necrotic cells were observed in theTaTLP1-OE line.We concluded that overexpression ofTaTLP1led to resistance to leaf rust.

The activities of antioxidant enzymes in inoculated leaves were measured.CAT activity continuously increased over 0–48 hpi and peaked at 96 hpi in bothTaTLP1-OE and JW1 plants.However,it was significantly higher at 96 and 120 hpi than that at 0,24 and 48 hpi inTaTLP1-OE plant.SOD activity inTaTLP1-OE and WT plants peaked at 24 hpi,and decreased and retained similar levels until 120 hpi(Fig.S3A).Fig.S3B showed that the expression of theTaSOD,TaCATandTaNOXgenes was induced following inoculation indicating that they participated in disease response.Expression levels ofTaCATin JW1 were significantly higher than those inTaTLP1-OE plants.BothTaSODandTaNOXgenes were upregulated in inoculatedTaTLP1-OE plants.Expression ofTaSODandTaNOXat four time points were higher than those in WT plants.These findings indicated that expression of ROS-related genes inTaTLP1-OE and JW1 plants were significantly different after inoculation,implying thatTaTLP1contributes to defense of wheat againstPtby regulating ROS accumulation.

3.3.Overexpression of TaTLP1 in wheat confers resistance to CRR

Three independentTaTLP1transgenic lines and JW1 as susceptible control were inoculated withB.sorokiniana.Reduced disease development was observed in theTaTLP1-OE lines at 3 dpi (days post inoculation).At 6 dpi brown necrotic lesions appeared at the stem base and roots of JW1 plants(Fig.3A–C;Table S5).Confocal microscopic examination of leaf cells showed delayed growth of infection hyphae and secondary hyphae,reduced hyphal length and area of infection inTaTLP1-OE leaves compared to JW1(Fig.3D).At 120 hpi,hyphal length and number of infection sites in infected leaves were significantly reduced inTaTLP1-OE plants compared to those in JW1 (Fig.3E,F).

Fig.1.Analysis of TaTLP1 expression and agronomic traits in TaTLP1-OE transgenic wheat.(A) Analysis of TaTLP1 expression in six T4 homozygous transgenic lines. TaTLP1 gene expression levels are normalized to that of GAPDH.Values are means of triplicate reactions of three independent biological samples.Significant differences are represented by asterisks and error bars represent the standard deviation.One-way ANOVA was used to test significance of differences.** and ***,significantly different at P<0.01 and P<0.001,respectively.(B)Phenotypic traits of JW1 and TaTLP1-OE transgenic plants.(1)Plant height.(2)Tiller number,spike morphology,and 1000 kernels.WT,JW1;OE,transgenic wheat line overexpressing TaTLP1.

Fig.2. TaTLP1-OE lines exhibit resistance to leaf rust.(A) Disease development on the TaTLP1-OE lines.WT,JW1;OE4–OE6,transgenic wheat lines overexpressing TaTLP1.Plants were spray-inoculated with Pt urediniospores and disease developments were monitored 14-days after inoculation.(B) H2O2 accumulation was visualised by DAB staining.Scale bars,20 μm.(C)Histology of hyphal development and host cell death during Pt infection.U,urediospore;GT,germ tube;A,appressorium;IH,infection hypha;HMC,haustorial mother cell;SH,secondary hypha;SV,substomatal vesicle;HR,hypersensitive reaction (green color).Scale bars,100 μm.(D) Measures of fungal development in infected OE and JW1 control plants (50 infection sites were scored per sample).One-way ANOVA was used to test the significance of differences.* and **,significantly different at P <0.05 and P <0.01,respectively.

Fig.3. TaTLP1-OE lines exhibit resistance to common root rot.(A)Disease development of common root rot in leaves and lower stem regions of JW1 and TaTLP1-OE lines 4–6.WT,JW1;OE4–OE6,transgenic wheat lines overexpressing TaTLP1.(B–F)Measures of root browning length(B);leaf necrosis area(C);Fungal spread in infected leaves(D);hyphal length (E);and infection area (F).One-way ANOVA was used to test the significance of difference.*,significantly different at P <0.05.All the data was measured by using image J software to quantify the disease development of CRR.IH,infection hypha;SH,secondary hypha.

POD and β-1,3-glucanase activity in JW1 plants after CRR inoculation remained at relatively similar levels at all time points.However,POD activity in theTaTLP1-OE lines increased over 0–48 hpi and peaked at 48 hpi,and then decreased (Fig.S4A).β-1,3-glucanase activity inTaTLP1-OE lines increased over 0–72 hpi and peaked at 72 hpi,and then decreased (Fig.S4B).The activity was significantly higher in theTaTLP1-OE lines than that in JW1 at any time point.In order to detect whetherTaTLP1expression can be induced byB.sorokinianaand related to resistance to root rot,TcLr19 as disease-resistant material was used to analyze the expression ofTaTLP1by qPCR.TaTLP1expression was strongly induced after CRR inoculation with TcLr19 and reached highest expression level at 72 hpi (Fig.S4C).The accumulated expression ofTaTLP1transcripts in infected TcLr19 plants was significantly higher than in the same genotype treated with water at 24,48,72 and 96 hpi.These observations indicated that overexpression ofTaTLP1enhanced resistance to CRR by increasing POD and β-1,3-glucanase activities.

4.Discussion

Previous studies showed that silencing ofTaTLP1in wheat line TcLr35 significantly reducedLr35-mediated resistance to leaf rust[8].In the present study,a wheat line withTaTLP1showed significant levels of resistance to CRR and leaf rust,indicating thatTaTLP1-OE lines had potential to be deployed to defence both pathogens in the field.

It has been documented that the antifungal activity of TLP protein is associated with its glucanase activity and the TLP proteins bind to and degrade β-1,3-glucanase the main component of fungal cell walls,thereby inhibiting the fungal growth [20].As we expected,the glucanase activity in theTaTLP1-OE plant was higher than that of WT plant after inoculation withB.sorokiniana(Fig.S4B).In addition to increased glucanase activity inTaTLP1-OE plants,we also observed that the activity of ROS-related enzyme POD increased in theTaTLP1-OE plant compared with that in the control plant at 48 hpi (Fig.S4A).These pieces of evidence strongly indicate that the enhanced resistance to root rot was directly associated with overexpression of theTaTLP1gene.To the best of our knowledge,this is the first time that transgenic wheat overexpressingTaTLP1was tested for response to CRR.However,based on these preliminary findings we cannot conclude that the molecular mechanisms underlyingTaTLP1-mediated resistance to leaf rust and CRR are the same.Future studies,such as identifying proteins interactingTaTLP1and downstream signaling pathways,are required to understand the function ofTaTLP1.

Previous studies have demonstrated that transgenic wheat lines overexpressing defense-related genes exhibit enhanced resistance to different fungal pathogens.For example,transgenic wheat overexpressing a defense response α-1-purothionin,TLPs or β-1,3-glucanase conferred significantly improved resistance to Fusarium head blight [21].Overexpression of a single PR gene or combination of two genes(chitinase and β-1,3-glucanase)in wheat resulted in partial resistance toFusarium graminearumunder greenhouse conditions,but was not effective in the field [22].These studies show that overexpression of a PR gene can achieve enhanced resistance to fungal pathogens.A recent study found that TaTLP1 interacted with TaPR1 to contribute to wheat defense responses to leaf rust[9].We speculate that TaTLP1 and TaPR1 genes coordinate defence responses.Generation of transgenic wheat cooverexpressingTaTLP1andTaPR1will be our next task to determine whether such a line has increased resistance to leaf rust and CRR compared with the line expressing onlyTaTLP1.It has also been documented thatTLP1plays a role in resistance to abiotic stress.For example,Onishi et al.[23] found that the soybean PR-5 gene (GmOLPa) was induced after treatment with 300 mm NaCl and acted as a protective PR-5 protein in the extracellular spaces of soybean roots in response to salt stress and dehydration.It will be very interesting to determine whether theTaTLP1-OE lines provide resistance to abiotic stresses.Although there no commercial transgenic wheat derived from overexpressing PR-related genes is available in the market the transgenic lines developed in this study provide potential for use of enhanced resistance to leaf rust and root rot in breeding.

CRediT authorship contribution statement

Zhongchi Cui:Conceptualization,Methodology,Data curation,Writing -original draft.Fang Liang:Methodology,Data curation.Jiarui Zhang:Data curation.Fei Wang:Methodology,Data curation.Daqun Liu:Conceptualization.Haiyan Wang:Conceptualization,Writing -original draft,Writing -review &editing.

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.

Acknowledgments

This work was supported by the Natural Science Foundation of Hebei(C2020204028)and the National Natural Science Foundation of China (31501623).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2021.03.021.