Overexpression of MdIPT8 Encodes an Isopentenyl Transferase Enzyme Enhances Resistance to Colletotrichum Gloeosporioides in apple (Malus Domestica)

doi:10.21203/rs.3.rs-721415/v1

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Overexpression of MdIPT8 Encodes an Isopentenyl Transferase Enzyme Enhances Resistance to Colletotrichum Gloeosporioides in apple (Malus Domestica)

https://doi.org/10.21203/rs.3.rs-721415/v1

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Apple (Malus domestica) is a delicious fruit and have high economic value. However, numerous destructive fungi affect apple tree and limit the development of apple production. Many researches showed that salicylic acid and jasmonic acid improved plant resistance to fungal disease, but the potential mechanisms between cytokinin and fungal disease were hardly reported. The IPT gene family encodes the proteins of synthetizing cytokinin and plays an indispensable role in plant biological stress. In our previous study, we found the disease resistance in autotetraploid apple was much better than ‘Hanfu’ and we analyze the gene expression in ‘Hanfu’ and autotetraploid apple. We found the MdIPT8 gene was significantly different expressed. Therefore, we focused on MdIPT genes. We uncovered ten IPT genes in the ‘Hanfu’ whole genome, that are unevenly distributed across five chromosomes. Phylogenetic analysis indicated this family falling into two groups. Moreover, the transcriptional analysis of ten IPT genes indicated MdIPT8 can be induced under Colletotrichum gloeosporioides stress stimulating in apple leaves. The MdIPT8 protein coalesced to green fluorescent protein pinpointed to the chloroplast. Finally, we confirmed that the overexpression of MdIPT8 increased resistance to C. gloeosporioides. Additionally, the endogenous cytokinin content of ‘Hanfu’ leaves increased under the stress of C. gloeosporioides and exogenous cytokinin improved the disease resistance of leaves. Our findings supply an overview of the identification of apple IPT gene family and increases the understanding of cytokinin in apple anthracnose resistance study.

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

Apple

Colletotrichum gloeosporioides

IPT gene family

Cytokinin

Expression analysis

We acquired the overexpression of MdIPT8 transgenic apple lines and confirmed they have stronger resistance to Colletotrichum. gloeosporioides.

Fruit tree yield is closely related to their growth and development. The pathogen resistance of apple trees determines the quality of fruits(Zhang et al. 2019b). When fungi attack apple trees, pathogens seriously hinder their growing and developing(He et al. 2018). C. gloeosporioides is a fungal pathogen that constitutes a serious threat to orchards occurring in tropical and subtropical(Moreira et al. 2021). Chemical treatments are effectively used to withhold fungal diseases(Gur et al. 2017, 2020; Pagès et al. 2020), but aforesaid treatments have negative side effects, for instance, environment unfriendly and food safety reduced(Hu et al. 2017). The most remarkable scenarios for conquering fungal diseases in apple are to select fungal resistant apple sources and cultivating fungal resistant variations through breeding(Chechi et al. 2019). As a result, it is very imperative to delve into the molecular mechanism potential fungal disease resistance in apple.

Plant defensed fungal disease in many pathways. Plant hormones play a meaningful role in plant fungal resistance signaling pathways(Pieterse et al. 2012). Large numbers of plant immune responses involving salicylic acid(Ding and Ding 2020), jasmonic acid(Zhao et al. 2019), and ethylene(Wang et al. 2020) have been reported. The effects of abscisic acid(Ha et al. 2012) and auxin(Jones et al. 2010) on plant resistance have also been gradually discovered. However, the influences of cytokinin to fungal disease were rarely reported.

Cytokinin, as one of the five important hormones in plants, is a decisive plant hormone that stimulate cell division and cell growth(Choi and Hwang 2007). It regulates plant physiology activities(Sakakibara 2006). It also affect plant disease resistance(Pertry et al. 2009). Researchers found that plant-derived cytokinin promotes Arabidopsis thaliana resistance to Pseudomonas syringae pv. Tomato DC3000 (Pst) and overexpression of genes encoding isopentenyl transferase enzyme (AtIPT1, 2, 5, and 7) raised transgenic Arabidopsis thaliana defense to Pst. In the interim, overexpression of cytokinin oxidase gene improved sensitivity of Arabidopsis thaliana to infection(Choi et al. 2010). The above outcomes indicated that cytokinin mediated plant to bacterial resistance. But the role of cytokinin in fungal disease resistance is still obscure.

Isopentenyl transferases (IPT) are key enzymes that catalyze the cytokinin biosynthesis and are also important rate-limiting enzymes that affect the types and levels of endogenous cytokinin in plants(Miyawaki et al. 2006). In plants, there are two types of isopentenyl transferase. One is the adenylate isopentenyl transferase (ATP/ADP-IPT) which the main active forms of biosynthesis of cytokinin are isopentenyl adenine and trans-zeatin (tZ). The other is tRNA-IPT, in which another active form involved in the catalytic synthesis of cytokinin is cis-zeatin (cZ). What’ s more, tZ was recognized to the most abundant and effective regulators in physiological processes(Hoyerová and Hošek 2020; Chen et al. 2021).

In our previous study, we found the expressed of IPT gene was significantly different between disease resistance autotetraploid apple and sensitive ‘Hanfu’(Ma et al. 2016). We confirmed that exogenous and endogenous cytokinin can increase apple leaves resistance to C. gloeosporioides. In this study, we singled out all potential IPT genes in the ‘Hanfu’ apple genome. Through bioinformatics analysis, physiological data analysis and transgenic technology to find the IPT gene which response to C. gloeosporioides. The results will contribute on apple resistance breeding.

2.1. Plant Materials

The material of plant tissue culture for ‘GL-3’, ‘Hanfu’ diploid and autotetraploid apple plants were grown at Shenyang Agriculture University (41.49°N, 123.34°E, Liaoning, China). The growth conditions in this study were described by Chen et al(Chen et al. 2019) and Zhang et al(Zhang et al. 2019a).

2.2. Fungal Culture and Infection

The fungus C. gloeosporioides performed for the experimentations were widened on potato dextrose agar cultures for a week about 28°C. Spores were gingerly placed in water. The suspension population was counted, then controlled to 10⁶ spores ml^− 1. The infected ‘HF’ plants were placed at 26°C for fungal development. At one day, two days, three days and four days after inoculation, the leaves were collected. They were frozen in pure nitrogen and transferred to -80℃.

2.3. Cytokinin Quantification and Treatments

For trans-zeatin (tZ) measurements, more 50 mg fresh weigh of leaf tissue was weighed. The samples were homogenized by tissue homogenizer below 0 ℃. The ratio of homogenization was 10%, which equivalent to 1 g tissue adding 9 ml homogenate. The homogenate was 0.01 M phosphate buffer (PH 7.4). The homogenized samples were centrifuged (5000 rpm, 15 min) and took the supernatant to measure. Cytokinin was measured using an ELISA kit specific for tZ (MEIMIAN, Jiangsu Meimian industrial Co., Ltd).

Thirty healthy diploid apple plants were sprayed with 0.02mg/ml tZ and immediately sprayed with 10⁸ spores ml^− 1 suspension. The inoculated plants were eventually incubated at 26°C before evaluation of disease. Three days and five days after inoculation, the leaves were observed and the disease indexes were calculated.

According to the percentage of observed the area of diseased leaf, the disease index of each leaf was rated. The disease conditions were graded as follows. 0 grade: no disease spots on leaves; 1 grade: the diseased leaf area accounts for less than 5% of the entire leaf area; 2 grade: the diseased leaf area accounts for 6%~10% of the entire leaf area; 3 grade: the diseased leaf area accounts for 11%~15% of the entire leaf area; 4 grade: the diseased leaf area accounts for 16%~20% of the entire leaf area; 5 grade: the diseased leaf area accounts for 21%~25% of the entire leaf area; 6 grade: the diseased leaf area accounts for 26%~30% of the entire leaf area; 7 grade: the diseased leaf area accounts for 31%~35% of the entire leaf area; 8 grade: the diseased leaf area accounts for 36%~40% of the entire leaf area; 9 grade: the diseased leaf area accounts for more than 40% of the entire leaf area.

$$\text{T}\text{h}\text{e} \text{d}\text{i}\text{s}\text{e}\text{a}\text{s}\text{e} \text{i}\text{n}\text{d}\text{e}\text{x} \left(\%\right)=\left[\sum \left(\text{A}\times \text{B}\right)÷\left(\text{C}\times \text{D}\right)\right]\times 100\%$$

A: Number of diseased leaves at all grade

B: Relative disease grade value

C: Total number of apple leaves

D: Highest disease grade value

2.4. Identification of IPT Gene Family

To identify IPT family genes in apple, BLAST algorithm-based searches were used to identify members of the IPT family. Arabidopsis thaliana and rice protein sequences were used to perform BLASTP algorithm-based searches against the GDR(Jung et al. 2019). Additionally, the sequences of Arabidopsis thaliana were procured from TAIR (https://www.arabidopsis.org/index.jsp). The rice sequences were downloaded from Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/index.shtml). Gene IDs and CDS of the MdIPT family were acquired from the GDR. The length of amino acid sequences of MdIPT proteins were obtained by a tool from the ExPasy website (http://web.expasy.org/protparam/).

2.5. Phylogenetic Analysis

For phylogenetic analysis, IPT orthologs in Arabidopsis thaliana and Rice were obtained at TAIR and RGAP. The protein sequences of ten MdIPTs, nine AtIPTs, and ten OsIPTs were aligned through ClustalW program using the format parameters at MEGA X. A phylogenetic tree was then established by using the neighbor-joining plan with 1000 bootstrap replicates(Kumar et al. 2018).

2.6. Chromosomal Locations

Information on the MdIPT genes, including chromosomal location and DNA sequences, was obtained from the GDR. MapInspect (https://www.plantbreeding.wur.nl/) was used to map the distribution of the MdIPT genes.

2.7. Total RNA Extraction and cDNA Synthesis

Total RNA were derived from apple tissues with the modified CTAB technique(Chang et al. 2007). The cDNAs were reverse transcribed by using the TaKaRa RT reagent kit and maintained at − 20℃ until use.

2.8. RT-qPCR Assays

The quantitative measurement of genes expression was performed on an ABI QuantStudio™12K Flex real-time PCR instrument. The reaction system and procedure refer to the instruction manual of SYBR Premix Ex Taq. The melting curve was analyzed and the related expression of genes were counted according to the 2^−ΔΔCt guideline after the reaction was completed. The apple EF-1a (Accession No. DQ341381) was used as a normalization for analysis. The primers used are filled in Supplemental Table.

2.9. Promoter Sequence Analysis of MdIPT Genes

Promoter sequences (2000 bp) of apple IPT genes were obtained from the GDR. They were analyzed in ten members of the MdIPT family by inputting the nucleic acid sequences in FASTA format into the Plant CARE, and the protocols were set to default(Lescot et al. 2002).

2.10. Subcellular Localization

The complete length MdIPT8 (MD13G1182800) cDNA were duplicated into the Nde Ⅰ and BamH Ⅰ sites in the pRI101-GFP to engender 35S::MdIPT8-GFP, and pRI101-GFP plasmid was used as a control. Different Agrobacterium solutions were transformed into Nicotiana benthamiana leaves. After 60 h of darkness, the signal of green fluorescent was scrutinized under a Leica TCS confocal microscope.

2.11. Construction of Expression Vector and Obtained Transgenic Plants

The full-length MdIPT8 cDNA were cloned and inserted into pRI101-AN binary vector plasmid so that MdIPT8 was under the regulation of 35S promotor. The gene was transformed into ‘GL-3’ based on the established protocol described by Chen et al(Chen et al. 2019).

2.12. Leaf Agroinfiltration and Fungal Inoculation

‘Hanfu’ diploid apple leaves were utilized for agroinfiltration, and Agrobacterium-attended transformation was executed by the injection progress(Meng et al. 2018). Two days after injection, spore suspension (10⁶/mL) of Colletotrichum gloeosporioides strain was cultivated on each site of injection after the apple leaves were disassembled, then they were hatched at 27 ℃ in damp dishes for 3 days in the darkness. Thirty leaves were used in each treatment. The ‘GL-3’ and overexpressing MdIPT8 lines were used for fungal inoculation. Spore suspension was injected to leaves in vitro and sprayed in the whole apple plants. After 3 and 5 days, they were observed.

3.1. Cytokinin Positively Regulates C. gloeosporioides Resistance in ‘Hanfu’ Apple

Previous research demonstrated that ‘Hanfu’ autotetraploid apple exhibited significantly differences in resistance to fungi, compared with ‘Hanfu’ apple(Chen et al. 2017) and cytokinin plays a momentous character in plant biotic stress(Choi et al. 2010). To determine the role of cytokinin in ‘Hanfu’ apple defense against C. gloeosporioides, we measured tZ content in ‘Hanfu’ and autotetraploid apple leaves. The concentration of tZ was significantly elevated in autotetraploid apple leaves compared to ‘Hanfu’ apple leaves (Figure. 1a). Subsequently, we also measured the tZ content of ‘Hanfu’ apple leaves in the C. gloeosporioides stress. We found that the concentration of tZ was elevated in the C. gloeosporioides treatment period (Figure. 1b). This observation suggests that cytokinin could regulate C. gloeosporioides resistance. To confirm this, we pre-treated the apple plants with exogenous cytokinin before infection with C. gloeosporioides. The results showed that exogenous cytokinin treatment significantly enhanced C. gloeosporioides resistance of the apple plants compared with the mock treatment (Figure. 1c, 1d). Taken together, cytokinin positively regulates C. gloeosporioides resistance in ‘Hanfu’ apple.

3.2. Identification of The IPT Gene Family in ‘Hanfu’ Apple

Previous research has shown that the cytokinin are mainly influenced by the balance between biosynthesis and catabolism. Isopentenyl transferases (IPT) are key enzymes that catalyze the cytokinin biosynthesis. In recent times, a high-quality assembly in ‘Hanfu’ were published(Zhang et al. 2019b). In this study, ten MdIPT genes (Table 1) were obtained using ‘HFTH1 Genome v1.0.a1 chromosomes’ database in GDR to examined the protein sequences of AtIPTs and OsIPTs. The IPT genes were named as MdIPT1 to MdIPT10, respectively, based on the chromosomal locations of the ‘Hanfu’ apple. The genomic distribution of chromosomal locations of MdIPT across seventeen chromosomal positions (Md01 − Md17) was figured out using MapInspect. A total of ten MdIPT genes were allotted to five chromosomal locations ranging from 1 to 3 genes per chromosome. Chromosomal location analyses showed that this family are dispersed on chromosome 16, chromosome 13, chromosome 11, chromosome 6 and chromosome 3 (Fig. S1). Chromosome 3 contains three MdIPT loci. Chromosome 6 contains one MdIPT loci. The rest chromosomes (Chr 11, 13 and 16) each harbors two loci. To validate the accuracy of the BLASTP results, these IPT family genes were further confirmed through BLASTN search at GenBank.

To understand and categorize the evolutionary relationships between MdIPTs and IPTs from other plants, a phylogenetic tree was organized using the amino acid sequences of ten MdIPTs, nine AtIPTs from Arabidopsis thaliana, and ten OsIPTs from rice (Fig. 2). This analysis indicated that apple IPTs could be grouped into two classifications based on information from Arabidopsis thaliana and rice. There are seven ATP/ADP-IPTs, including MdIPT1, MdIPT3, MdIPT6, MdIPT7, MdIPT8, MdIPT9 and MdIPT10, and three tRNA-IPTs, including MdIPT1, MdIPT2 and MdIPT5. By comparing the evolution of the three plants, interesting features were identified. All IPT homologs showed similar clustering patterns among Arabidopsis thaliana, rice, and apple.

Table 1

*MdIPT* gene family
Gene name	Gene ID	CDS length(bp)	Protein length(aa)
MdIPT1	HF40675-RA	1551	516
MdIPT2	HF03183-RA	1350	449
MdIPT3	HF02931-RA	960	320
MdIPT4	HF37306-RA	876	291
MdIPT5	HF28031-RA	2841	946
MdIPT6	HF28291-RA	897	298
MdIPT7	HF42175-RA	948	315
MdIPT8	Not Given	1011	336
MdIPT9	HF07762-RA	948	315
MdIPT10	HF13830-RA	1245	414

3.3. MdIPT8 Act as A C. gloeosporioides Responsive Gene

We found that C. gloeosporioides can induce apple to produce cytokinin (Fig. 1b) and exogenous cytokinin may reduce disease incidence (Fig. 1c). Thus, an application of C. gloeosporioides was further investigated the expression of MdIPT genes. As shown in Fig. 3, eight genes (MdIPT3, MdIPT4, MdIPT5, MdIPT6, MdIPT7, MdIPT8, MdIPT9, and MdIPT10) were unquestionably up-regulated at 2 days or 3 days after inoculating C. gloeosporioides. On the contrary, MdIPT2, MdIPT3 and MdIPT6 were distinctly down-regulated at 1 day after C. gloeosporioides treatment. The expression of MdIPT3 and MdIPT8 gene increased in response to C. gloeosporioides treatment until 3 days. In contrast, MdIPT2 were considerably down-regulated after fungal treatment. MdIPT8 was up-regulated after C. gloeosporioides application at all subsequent time points. As a result, MdIPT8 act as a C. gloeosporioides responsive gene. Therefore, we focused our analysis on MdIPT8.

3.4. Multiple Organs Expression Analysis of MdIPT8 and Subcellular Localization of MdIPT8

‘Hanfu’ apple tissues were applied to recognize the expression levels of MdIPT8 by real-time polymerase chain reaction (Fig. S2). MdIPT8 was probed in all tissues, including root, fruit, stem, leaf, and flower. In the leaf, the highest expression level was spotted (Fig. S2).

To examine the subcellular localization of MdIPT8, we impermanently expressed MdIPT8-GFP constructs in Nicotiana benthamiana leaves. These synthesis protein signals were overseen by Leica microscopy. Green brightness was apparently monitored in the chloroplast (Fig. 4). But, in the control group, the luminance signal of GFP was observed all over the tobacco cell (Fig. 4).

3.5. MdIPT8 Positively Regulates C. gloeosporioides Resistance in Apple

For further analysis, we cloned MdIPT8 by using PCR. To determine if MdIPT8 works in defense against C. gloeosporioides in apple, a 35S::MdIPT8 vector was transiently expressed in apple leaves via agroinfiltration, followed by inoculation with C. gloeosporioides (Fig. 7b). After inoculation 3 days, necrosis was monitored round the injection site on leaves (Fig. 7c). Overexpression of MdIPT8 decreased the area of necrosis, demonstrating that MdIPT8 gene enables reduce the fungus of virulence to apple leaves (Fig. 7c, 7d). In summary, these results indicated that MdIPT8 positively regulates C. gloeosporioides resistance in apple.

To preliminarily explore how IPT genes are regulated, a 2 kb promoter region for all IPT genes was identified, and the Plant CARE website was used to identify cis-elements. The promotor sequences of MdIPTs was accessed through the JBrowse tool in GDR. The prediction results show that the promoters of IPT genes contain multiple regulatory elements: phytohormone responses elements (ERE, TGA-element, ABRE, AuxRR-core, P-box, TATC-element, etc.), stress responses elements (DRE1, G-box, ARE, W-box, TGACG-motif, GC-motif, etc.), tissue-specific expression elements (CAT-box, CCGTCC motif) and light responsiveness elements (I-box, ACE, ATCT-motif) (Fig. S3). Most genes in the family can participate in stress resistances and are regulated by other plant hormones. Of the family, ten genes are ubiquitously controlled by stress responses.

3.6 Overexpression MdIPT8 improves resistance to Colletotrichum gloeosporioides in transgenic apple

To elucidate the role of MdIPT8 in C. gloeosporioides resistance, we overexpressed MdIPT8 in ‘GL-3’ using Agrobacterium-mediated method. We created three MdIPT8-OE lines. Then, we identified three putative transgenic plants by qRT-PCR. Apple RNA were extracted from ‘GL-3’ (WT) and three independent MdIPT8 transgenic lines for quantitative real-time PCR. Analysis of MdIPT8 expression levels showed that the expressing level of MdIPT8 in three transgenic lines were significantly higher than in WT (Fig. 6a). MdIPT8-OE line #3 have highest expression levels, and the relative expression levels were 14.9-flod higher than they were in the ‘GL-3’ plants. Meanwhile, we measured tZ content in two high expressing level lines and tZ content in transgenic plants were higher than in WT plants (Fig. 6b). Thus, we obtained overexpressing MdIPT8 apple plants.

To characterize the role of MdIPT8 in defense against C. gloeosporioides in apple, we tested the effects of C. gloeosporioides to ‘GL-3’, MdIPT8-OE line #2 and MdIPT8-OE line #3 plants. Treatment with C. gloeosporioides for 3 days and 5 days accelerated diseased areas development in ‘GL-3’ leaves and plants, but only slightly influenced infected areas in overexpression MdIPT8 leaves and plants (Fig. 7a and c). Analysis of disease index showed that apple transgenic plants to C. gloeosporioides resistance were much better than WT palnts (Fig. 7b and d).

In fruit trees, apple is one of the most influential economic value(Zhang et al. 2019b). Nevertheless, apple production is diminished via many kinds of fungal diseases(He et al. 2018). Apple anthracnose, caused by Colletotrichum spp., causes significant yield losses to the growers(Moreira et al. 2021). Therefore, analysis of the apple response mechanism is of great significance for increasing apple yield. Some plant hormones (such as jasmonate and abscisic acid)(Dugé De Bernonville et al. 2012; Su et al. 2020) can activate the transcription of many defense-related genes and help regulate plant stress responses. A few reports scrutinized the roles of cytokinin against fungal disease. Overall, the mechanism by which cytokinin regulates apple fungal stress is poorly understood. Here, we found that exogenous spraying cytokinin increased apple resistance to C. gloeosporioides (Fig. 1c). Endogenous cytokinin content were significant higher in the C. gloeosporioides treated plants than in the untreated plants and showed a positive correlation (Fig. 1b). The results show that cytokinin improve apple disease resistance.

IPT genes are cytokinin biosynthesis genes. In plants, it play a paramount function in fine-turning cytokinin(Ha et al. 2012). Previously, plentiful studies have analyzed the functional of IPT genes in the model plants(Miyawaki et al. 2004) and non-model plant(Žižková et al. 2015) using forward techniques of bioinformatics. AtIPT genes have been more functionally verified(Galichet et al. 2008). In Arabidopsis, the transgenic plants enhance expression of AtIPT genes, and contain more sufficient cytokinin(Guo and Gan 2011). The overexpression of MdIPT3a inhibits root development, a delay in flowering, and enhanced outgrowth of axillary buds(Zhu et al. 2012). However, previous research results mostly focused on how IPT affects plant growth and development, and there are few reports on the regulation processes involved in fungal response. In order to acquire information about MdIPT potential function after inoculating C. gloeosporioides, we kept a genetic expression experiment to analyze ‘Hanfu’ IPT family members. Thus, we found that the expression of MdIPT8 can be activated by fungus treatments (Fig. 3), supposing that MdIPT8 may be involved in hormone-induced apple growing and developing even respond to various pathogen stresses (Figs. 5 and 7). This is basically consistent with the results of previous research reports that its homologous gene AtIPT1 can significantly respond to pathogen stress in Arabidopsis(Choi et al. 2010). All in all, this research disclosed a novel mechanism wherein cytokinin regulate fungal stress tolerance in apple by the expression of MdIPT8.

Promoter sequence analysis disclosed the presence of many cis-acting elements correlated with hormone and stress responses in the MdIPT gene promoter (Fig. S3). In other researcher’s study, MdIPT5b expression maintains high cytokinin levels, leading to improve salt tolerance(Feng et al. 2019). Therefore, some abiotic stress response factors may be critical for regulating the expression of MdIPT and speculate that biological stress may also play a fundamental role in conducting MdIPT genes expression. WRKY is one of the most enormous transcription factor families. The proteins of WRKY family are relevant with many fungal stress defense pathways. WRKY proteins can function due to the highly conserved WRKY domain that binds the W-box region(Rushton et al. 2010). The 2000bp promoter region of MdIPT8 have W-box (Fig. S3), so we suggested that the expression of MdIPT8 probably is regulate by WRKY proteins so that MdIPT8 enhance the resistance of apple fungal disease.

Hence, our research demonstrated that MdIPT8 regulates C. gloeosporioides stress in apple by improving cytokinin content. It was the first to report the cytokinin response to C. gloeosporioides in apple by molecular methods. Our findings provide genetic resources and theoretical bases for the molecular breeding of fungal stress in apple.

Funding

This work was supported by the National Key R&D Program of China (No. 2019YFD1000100), the Nature Science Project of the Technology Department of Liaoning Province (No. 20180550478 and No. 2020-MS-198), the Millions Talents Project Foundation of Liaoning Province (No. 2017069), the LiaoNing Revitalization Talents Program (No. XLYC1902069), Shenyang Young and Middle-aged Science and Technology Innovation Talents Support Plan (RC190446).

Conflict of interests

The authors declare that they have no conflicts of interest to report.

Author contributions

Conceptualization: Yue Ma and Feng Wang, Funding acquisition: Yue Ma and Zhigang Wang, Formal analysis: Jiajun Shi and Feng Zhang, Project administration: Yue Ma and Jiajun Shi, Resources: Yue Ma and Feng Wang, Supervision: Yue Ma and Feng Wang, Validation: Feng Wang, Jiajun Shi, Feng Zhang, Qiu Jiang, Yuhong Yuan, Xiaolin Nie and Ying Zhou, Visualization: Jiajun Shi and Feng Zhang, Supervision: Yue Ma and Feng Wang, Writing - original draft: Jiajun Shi and Feng Zhang, Writing - review & editing: Yue Ma and Jiajun Shi.

Chang L, Zhang Z, Yang H et al (2007) Detection of Strawberry RNA and DNA Viruses by RT-PCR Using Total Nucleic Acid as a Template. J Phytopathol 155:431–436. https://doi.org/10.1111/j.1439-0434.2007.01254.x
Chechi A, Stahlecker J, Dowling ME, Schnabel G (2019) Diversity in species composition and fungicide resistance profiles in Colletotrichum isolates from apples. Pestic Biochem Physiol 158:18–24. https://doi.org/10.1016/j.pestbp.2019.04.002
Chen K, Song M, Guo Y et al (2019) MdMYB46 could enhance salt and osmotic stress tolerance in apple by directly activating stress-responsive signals. Plant Biotechnol J 17:2341–2355. https://doi.org/10.1111/pbi.13151
Chen L, Zhao J, Song J, Jameson PE (2021) Cytokinin glucosyl transferases, key regulators of cytokinin homeostasis, have potential value for wheat improvement. Plant Biotechnol J 19:878–896. https://doi.org/10.1111/pbi.13595
Chen M, Wang F, Zhang Z et al (2017) Characterization of fungi resistance in two autotetraploid apple cultivars. Sci Hortic 220:27–35. https://doi.org/10.1016/j.scienta.2017.03.034
Choi J, Huh SU, Kojima M et al (2010) The Cytokinin-Activated Transcription Factor ARR2 Promotes Plant Immunity via TGA3/NPR1-Dependent Salicylic Acid Signaling in Arabidopsis. Dev Cell 19:284–295. https://doi.org/10.1016/j.devcel.2010.07.011
Choi J, Hwang I (2007) Cytokinin: perception, signal transduction, and role in plant growth and development. J Plant Biol 50:98–108. https://doi.org/10.1007/BF03030617
Ding P, Ding Y (2020) Stories of Salicylic Acid: A Plant Defense Hormone. Trends Plant Sci 25:549–565. https://doi.org/10.1016/j.tplants.2020.01.004
Dugé De Bernonville T, Gaucher M, Flors V et al (2012) T3SS-dependent differential modulations of the jasmonic acid pathway in susceptible and resistant genotypes of Malus spp. challenged with Erwinia amylovora. Plant Sci 188–189:1–9. https://doi.org/10.1016/j.plantsci.2012.02.009
Feng Y, Liu J, Zhai L et al (2019) Natural variation in cytokinin maintenance improves salt tolerance in apple rootstocks. Plant Cell Environ 42:424–436. https://doi.org/10.1111/pce.13403
Galichet A, Hoyerová K, Kamínek M, Gruissem W (2008) Farnesylation directs AtIPT3 subcellular localization and modulates cytokinin biosynthesis in Arabidopsis. Plant Physiol 146:1155–1164. https://doi.org/10.1104/pp.107.107425
Guo Y, Gan S (2011) AtMYB2 regulates whole plant senescence by inhibiting cytokinin-mediated branching at late stages of development in Arabidopsis. Plant Physiol 156:1612–1619. https://doi.org/10.1104/pp.111.177022
Gur L, Reuveni M, Cohen Y (2020) Control of Alternaria fruit rot in “Pink Lady” apples by fungicidal mixtures. Crop Prot 127:104947. https://doi.org/10.1016/j.cropro.2019.104947
Gur L, Reuveni M, Cohen Y (2017) Phenology-Based Management of Alternaria Fruit Rot in Pink Lady Apples. Plant Dis 102:1072–1080. https://doi.org/10.1094/PDIS-05-17-0735-RE
Ha S, Vankova R, Yamaguchi-Shinozaki K et al (2012) Cytokinins: metabolism and function in plant adaptation to environmental stresses. Trends Plant Sci 17:172–179. https://doi.org/10.1016/j.tplants.2011.12.005
He L, Li X, Gao Y et al (2018) Characterization and Fungicide Sensitivity of Colletotrichum spp. from Different Hosts in Shandong, China. Plant Dis 103:34–43. https://doi.org/10.1094/PDIS-04-18-0597-RE
Hoyerová K, Hošek P (2020) New Insights Into the Metabolism and Role of Cytokinin N-Glucosides in Plants. Front Plant Sci 11:741. https://doi.org/10.3389/fpls.2020.00741
Hu X, Zhong Y, Huang K et al (2017) Differential expression of 12 NBS-encoding genes in two apple cultivars in response to Alternaria alternata f. sp. mali infection. Can J Plant Sci 98:279–287. https://doi.org/10.1139/cjps-2017-0117
Jones B, Gunnerås SA, Petersson SV et al (2010) Cytokinin regulation of auxin synthesis in Arabidopsis involves a homeostatic feedback loop regulated via auxin and cytokinin signal transduction. Plant Cell 22:2956–2969. https://doi.org/10.1105/tpc.110.074856
Jung S, Lee T, Cheng C-H et al (2019) 15 years of GDR: New data and functionality in the Genome Database for Rosaceae. Nucleic Acids Res 47:D1137–D1145. https://doi.org/10.1093/nar/gky1000
Kumar S, Stecher G, Li M et al (2018) MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy096
Lescot M, Déhais P, Thijs G et al (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30:325–327. https://doi.org/10.1093/nar/30.1.325
Ma Y, Xue H, Zhang L et al (2016) Involvement of Auxin and Brassinosteroid in Dwarfism of Autotetraploid Apple (Malus × domestica). Sci Rep 6:26719–26719. https://doi.org/10.1038/srep26719
Meng D, Li C, Park H-J et al (2018) Sorbitol Modulates Resistance to Alternaria alternata by Regulating the Expression of an NLR Resistance Gene in Apple. Plant Cell 30:1562–1581. https://doi.org/10.1105/tpc.18.00231
Miyawaki K, Matsumoto-Kitano M, Kakimoto T (2004) Expression of cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis: tissue specificity and regulation by auxin, cytokinin, and nitrate. Plant J 37:128–138. https://doi.org/10.1046/j.1365-313X.2003.01945.x
Miyawaki K, Tarkowski P, Matsumoto-Kitano M et al (2006) Roles of Arabidopsis ATP/ADP isopentenyltransferases and tRNA isopentenyltransferases in cytokinin biosynthesis. Proc Natl Acad Sci U S A 103:16598–16603. https://doi.org/10.1073/pnas.0603522103
Moreira RR, Zielinski EC, Castellar C et al (2021) Study of infection process of five species of Colletotrichum comparing symptoms of glomerella leaf spot and bitter rot in two apple cultivars. Eur J Plant Pathol 159:37–53. https://doi.org/10.1007/s10658-020-02138-y
Pagès M, Kleiber D, Violleau F (2020) Ozonation of three different fungal conidia associated with apple disease: Importance of spore surface and membrane phospholipid oxidation. Food Sci Nutr 8:5292–5297. https://doi.org/10.1002/fsn3.1618
Pertry I, Václavíková K, Depuydt S et al (2009) Identification of Rhodococcus fascians cytokinins and their modus operandi to reshape the plant. Proc Natl Acad Sci U S A 106:929–934. https://doi.org/10.1073/pnas.0811683106
Pieterse CMJ, Van der Does D, Zamioudis C et al (2012) Hormonal Modulation of Plant Immunity. Annu Rev Cell Dev Biol 28:489–521. https://doi.org/10.1146/annurev-cellbio-092910-154055
Rushton PJ, Somssich IE, Ringler P, Shen QJ (2010) WRKY transcription factors. Trends Plant Sci 15:247–258. https://doi.org/10.1016/j.tplants.2010.02.006
Sakakibara H (2006) CYTOKININS: Activity, Biosynthesis, and Translocation. Annu Rev Plant Biol 57:431–449. https://doi.org/10.1146/annurev.arplant.57.032905.105231
Su W, Huang L, Ling H et al (2020) Sugarcane calcineurin B-like (CBL) genes play important but versatile roles in regulation of responses to biotic and abiotic stresses. Sci Rep 10:167–167. https://doi.org/10.1038/s41598-019-57058-7
Wang J-H, Gu K-D, Han P-L et al (2020) Apple ethylene response factor MdERF11 confers resistance to fungal pathogen Botryosphaeria dothidea. Plant Sci 291:110351. https://doi.org/10.1016/j.plantsci.2019.110351
Zhang F, Wang F, Yang S et al (2019a) MdWRKY100 encodes a group I WRKY transcription factor in Malus domestica that positively regulates resistance to Colletotrichum gloeosporioides infection. Plant Sci 286:68–77. https://doi.org/10.1016/j.plantsci.2019.06.001
Zhang L, Hu J, Han X et al (2019b) A high-quality apple genome assembly reveals the association of a retrotransposon and red fruit colour. Nat Commun 10:1494–1494. https://doi.org/10.1038/s41467-019-09518-x
Zhao X-Y, Qi C-H, Jiang H et al (2019) Functional identification of apple on MdHIR4 in biotic stress. Plant Sci 283:396–406. https://doi.org/10.1016/j.plantsci.2018.10.023
Zhu YD, Jin YS, Wei S et al (2012) Functional analysis of the isopentenyltransferase gene MdIPT3a from apple (Malus pumila Mill.). J Hortic Sci Biotechnol 87:478–484. https://doi.org/10.1080/14620316.2012.11512898
Žižková E, Dobrev PI, Muhovski Y et al (2015) Tomato (Solanum lycopersicum L.) SlIPT3 and SlIPT4 isopentenyltransferases mediate salt stress response in tomato. BMC Plant Biol 15:85–85. https://doi.org/10.1186/s12870-015-0415-7

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Overexpression of MdIPT8 Encodes an Isopentenyl Transferase Enzyme Enhances Resistance to Colletotrichum Gloeosporioides in apple (Malus Domestica)

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Abstract

Figures

Key Message

1. Introduction

2. Materials And Methods

2.1. Plant Materials

2.2. Fungal Culture and Infection

2.3. Cytokinin Quantification and Treatments

2.4. Identification of IPT Gene Family

2.5. Phylogenetic Analysis

2.6. Chromosomal Locations

2.7. Total RNA Extraction and cDNA Synthesis

2.8. RT-qPCR Assays

2.9. Promoter Sequence Analysis of MdIPT Genes

2.10. Subcellular Localization

2.11. Construction of Expression Vector and Obtained Transgenic Plants

2.12. Leaf Agroinfiltration and Fungal Inoculation

3. Results

3.1. Cytokinin Positively Regulates C. gloeosporioides Resistance in ‘Hanfu’ Apple

3.2. Identification of The IPT Gene Family in ‘Hanfu’ Apple

3.3. MdIPT8 Act as A C. gloeosporioides Responsive Gene

3.4. Multiple Organs Expression Analysis of MdIPT8 and Subcellular Localization of MdIPT8

3.5. MdIPT8 Positively Regulates C. gloeosporioides Resistance in Apple

3.6 Overexpression MdIPT8 improves resistance to Colletotrichum gloeosporioides in transgenic apple

4. Discussion

Declarations

References

Supplementary Files

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