Next Article in Journal
Changes in the Composition of Flavonols and Organic Acids during Ripening for Three cv. Sauvignon Blanc Clones Grown in a Cool-Climate Valley
Next Article in Special Issue
Validation of Propidium Monoazide-qPCR for Assessing Treatment Effectiveness against ‘Candidatus Liberibacter asiaticus’ in Citrus
Previous Article in Journal
A New Strategy to Improve Vineyard Resilience: Grapevine Morphological Adaptation to Short-Term Nitrogen Deficiency
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 12
Issue 6
10.3390/agronomy12061356
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Key Role of Heat Shock Protein Expression Induced by Ampicillin in Citrus Defense against Huanglongbing: A Transcriptomics Study

by
Chuanyu Yang
1,2,*,
Charles Powell
1,
Yongping Duan
3,
Xiongjie Lin
4,
Goucheng Fan
5,
Hanqing Hu
4,* and
Muqing Zhang
1,6
1
IRREC-IFAS, University of Florida, 2199 S. Rock Rd, Fort Pierce, FL 34945, USA
2
Citrus Center, Texas A&M University-Kingsville, 312 N. International Blvd, Weslaco, TX 785799, USA
3
USHRL-USDA-ARS, 2011 S. Rock Rd, Fort Pierce, FL 34945, USA
4
Institute of Fruit Tree Research, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China
5
Institute of Plant Protection, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China
6
State Key Lab for Conservation and Utilization of Subtropical Agri-Biological Resources, Guangxi University, Nanning 530005, China
*
Authors to whom correspondence should be addressed.
Agronomy 2022, 12(6), 1356; https://doi.org/10.3390/agronomy12061356
Submission received: 17 March 2022 / Revised: 25 May 2022 / Accepted: 29 May 2022 / Published: 2 June 2022
(This article belongs to the Special Issue Citrus Bacterial Diseases Spread and Control Strategies in the World: Management and Molecular Approaches)
Browse Figures
Review Reports Versions Notes

Abstract

:
Citrus Huanglongbing (HLB) is a serious disease for the citrus industry. Earlier studies showed that ampicillin (Amp) can reduce titers of the pathogen which causes HLB and the bacteria Candidatus Liberibacter asiaticus (CLas) in HLB-affected citrus. CLas has not yet been cultured, so the mechanisms of Amp against CLas are unclear. Some chemicals were demonstrated to trigger citrus defense systems against CLas. Therefore, we hypothesize that Amp may induce citrus defenses against CLas. Here we applied three nano-formulations of varying droplet sizes to HLB-affected citrus to achieve different accumulated concentrations of Amp (high, medium, low) in the plants. We then used RNA-seq to analyze induction of gene expression of citrus defense systems against CLas in response to different concentrations of Amp. The results indicated that at all accumulated concentrations of Amp can significantly suppress CLas titer and mitigate HLB symptoms. Transcriptomic analyses showed that Amp treatment induced expression of heat shock proteins (Hsps) in HLB-affected citrus, and these Hsps were significantly related to several defense genes encoding R proteins, transcription factors, splicing factors, RNA-binding proteins, RNA-dependent RNA polymerase, Gibberellic acid methyltransferase 2, L-ascorbate peroxidase 2, and ferruginol synthase that confer tolerance to CLas in citrus plants. Taken together, these results suggest that Amp treatment of citrus plants can trigger expression of Hsps and related defense genes to respond to CLas infection. These findings are valuable for developing novel strategies to combat citrus HLB.

1. Introduction

Citrus Huanglongbing (HLB) is a catastrophic disease that threatens the citrus industry worldwide. HLB is caused by the psyllid-transmitted, phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) [1,2,3]. Florida is the main site of citrus production in the United States. Since HLB was first found in Florida in 2005 [4], citrus acreage and yield in Florida has decreased by 38% and 74%, respectively [5]. Sweet orange production dropped from 150 million boxes in 2005–2006 to 53 million boxes in 2020–2021 [5,6,7,8]. HLB symptomology includes yellowing of shoots, blotchy mottled leaves, corky veins, malformed and discolored fruits, premature fruit drop, root loss, and eventually tree death [9,10]. To date, there are no effective management methods for infected trees in field, and there are no resistant commercial citrus cultivars [1].
Defense systems in plants can be activated by pathogens [11], beneficial microorganisms [12], and chemicals [13]. Plants can be induced to develop enhanced resistance to pathogen infection by treatment with numbers of inducers. Resistance induced by these agents is broad spectrum and long-lasting, but rarely provides complete control of infection. In fact, many resistance inducers provide between 20 to 85% disease control [13,14].
Multiple chemicals can induce citrus defense systems against CLas and alleviate HLB symptoms [15,16,17,18]. For example, several systemic acquired resistance (SAR) activators, which can enhance expression of pathogenesis-related (PR) genes, provide significant control of HLB by reducing CLas titer and disease progression when applied via trunk injection [15]. Sulphonamide antibiotics can induce expression of citrus genes related to the metabolism of jasmonates, brassinosteroids, reactive oxygen species, and secondary metabolites, which are all beneficial for HLB tolerance [18]. Elucidating the mechanism of citrus defense pathways triggered by inducers can be valuable for developing effective strategies to control citrus HLB.
Previous studies demonstrated that Ampicillin (Amp) can reduce CLas titer and mitigate HLB symptoms in HLB-affected citrus [19,20]. Amp is a Beta-lactam antibiotic that inhibits the growth of sensitive bacteria by inactivating enzymes located in the bacterial cell membrane, known as penicillin binding proteins, which are involved in cell wall synthesis [21]. In addition to this bactericidal activity, Amp treatment of HLB-affected citrus promotes plant growth [22,23] and enriches the population of beneficial bacteria that can induce pathogen defense pathways [24,25]. However, since CLas has not yet been cultured, whether Amp can directly inhibit CLas or promote plant growth is still unknown.
To date, no studies have elucidated the mechanisms of Amp against CLas in HLB-affected citrus. Our previous study showed that a nano-emulsion system is an efficient method for delivering chemical compounds into citrus plants, and that the accumulated concentration of Amp in HLB-affected citrus differed significantly depending on the droplet size of nano-emulsion coupled with Amp that was applied [26]. Here, we hypothesize that Amp may induce citrus defense against CLas. In this study, we applied nano-emulsions coupled with Amp that had different droplet sizes to HLB-affected citrus. We then used RNA-seq to examine changes in gene expression to identify which pathways are altered by different concentrations of Amp in HLB-affected citrus.

2. Materials and Methods

2.1. Plant Materials

Two-year-old healthy grapefruit (Citrus paradisi) seedlings were graft-inoculated (bark-grafting) with scions from HLB-affected lemon (C. limon) and subsequently maintained in a greenhouse at 25–28 °C, 60–70% relative humidity (RH), and irrigated three times a week. After 10 months, typical HLB symptoms such as vein corking and blotchy mottles appeared on the leaves of the inoculated seedlings. HLB-affected citrus seedlings exhibiting typical HLB symptoms were then detected for the presence of CLas bacteria by quantitative real-time polymerase chain reaction (RT-qPCR) using CLas-specific primers (HLBas, HLBr, and HLBp) [27].

2.2. Application of Oil in Water (O/W) Nano-Formulation to HLB-Affected Citrus in a Greenhouse

Oil in water (O/W) nano-emulsions were prepared using a spontaneous emulsification method as we previously described [26]. Following this protocol, two nano-emulsions: Nano-Cre and Nano-Soy, were prepared using Cremophor EL and soybean oil, respectively. The prepared nano-emulsions were then dissolved into a 3000 mg/L ampicillin solution (water containing 0.1% Brij35) at a ratio of 1:50 g/mL (w/v) to yield the nano-formulations Nano-Cre-Amp and Nano-Soy-Amp. The final ampicillin concentration of the nano-formulations (Nano-Cre-Amp and Nano-Soy-Amp) was 3000 mg/L. Ampicillin was also dissolved in water at a final concentration of 3000 mg/L as a negative control (Amp solution). Tap water containing 0.1% Brij 35 served as the blank control.
Droplet sizes in Nano-Cre-Amp, Nano-Soy-Amp, Amp solution, and Blank control (CK) were determined using a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, U.K.). The nano-formulations and control solutions were then applied to HLB-affected citrus by bark application with six replicates each. In 2-year-old HLB citrus trees, a sharp knife was used to scrape a very thin layer of bark from a 10 cm section of trunk, which was then wrapped with a 24 × 12 cm sponge (C31 large commercial sponge), to which 500 mL of nano-formulation or Amp solution was applied. On 4 days after the initial treatment, leaves were collected for bioassays to detect Amp concentration and to extract RNA for transcriptomic analysis.

2.3. Bioassay Method to Detect Amp

The bioassay used to detect accumulated Amp in citrus plants was previously described [26]. The zone diameter was indicative of the amount of ampicillin in the leaf extract during the experimental period. The Amp content was calculated using the formula (Log y = 5.12x, R2 = 0.99), where x is the Amp content in ng and y is the diameter of inhibition zone in cm.

2.4. RNA Isolation, cDNA Library Construction, Sequencing, De Novo Assembly, Quantifying Gene Expression, and RT-qPCR Analysis for Gene Expression Validation

Total RNA was isolated from three washed citrus leaves (200 mg) using a RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA), following the manufacturer’s instructions. Complete DNA removal was obtained through on-column DNase I treatment using a RNase-Free DNase Set (Qiagen, Valencia, CA, USA). The quantity and purity of RNA was evaluated using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Equal amounts of total RNA from three biological replicates were pooled for Illumina deep RNA sequencing, in order to improve reliability and decrease the likelihood of biological error. The quality of the library was assessed using an Agilent Bioanalyzer 2100 system. For library assembly, adapter sequences and low-quality reads (base quality < 20; read length < 40 bp) were discarded from the raw reads.
The high-quality reads were mapped to the Citrus sinensis reference genome sequence using Bowtie and BWA [28,29,30]. Levels of Gene expression were calculated as reads per kilobase of exon model per million mapped reads (RPKM) [31]. Sets of differentially expressed genes (DEGs) were identified using edgeR with a |Log2(fold-change)| > 1.0 and an FDR ≤ 0.05. Clustered profiles with p-value ≤ 0.05 were considered to be significantly expressed. Gene Ontology (GO) annotation was conducted using Blast2 GO software [32]. The GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were searched with the enriched DEGs to identify changes in biological functions and metabolic pathways.
Quantitative real-time PCR (RT-qPCR) was conducted using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) to examine expression patterns of 15 selected DEGs. Specific primers for the DEGs were designed using Primer 3.0 [33] and are listed in Supplementary Table S1. The citrus GAPDH gene was selected as an internal control to normalize expression levels of the target genes among different samples. RT-qPCR was performed with three biological repetitions and three technical repetitions per each biological repetition. All RT-qPCR reactions were carried out in 96-well plates. RT-qPCR was performed in a 20 µL reaction volume that contained 5 µL Thunderbird TM SYBR qPCR Mix (TOYOBO, Osaka, Japan), 1 µL cDNA, 1 mM gene-specific primers, and 3 µL ddH2O. To analyze dissociation curve profiles, the following program was run after the 40 cycles of PCR: 95 °C for 15 s, followed by a constant increase in temperature between 60 °C and 95 °C. Expression was calculated by 2−ΔΔCt and normalized against level of GAPDH gene expression [34].

2.5. Genomic DNA isolation and CLas Detection

To detect CLas titers after nano-formulation treatments, three HLB symptom leaves were collected from treated citrus 0, 120, and 240 days after the initial treatment. Each leaf was rinsed three times with sterile water. Midribs were separated from the leaf samples and cut into 1.0 to 2.0 mm-long pieces. DNA was isolated from 0.1 g (fresh weight) of leaf midrib tissue using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. qPCR was conducted with primers and probes (HLBas, HLBr and HLBp) [27] for CLas using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) in a 20 μL reaction volume containing 300 nM (each) target primer (HLBas and HLBr), 150 nM target probe (HLBp), and 1× TaqMan qPCR Mix (Applied Biosystems). The amplification protocol was 95 °C for 20 s followed by 40 cycles at 95 °C for 3 s and 60 °C for 30 s. All reactions were performed in triplicate and each run contained one negative (DNA from a healthy grapefruit) and one positive (DNA from a CLas-infected grapefruit) control. Data were analyzed using the ABI 7500 Fast Real-Time PCR System with SDS software. The resulting Ct values were converted to estimated bacterial population by the regression equation Y = 13.82 − 0.2866X, where Y is the estimated log concentration of template and X is the Ct value from qPCR [27,35].

2.6. Data Analysis

Differences among different treatments including different nano-formulations and accumulated concentrations of Amp, were assessed by Duncan’s multiple range tests at p < 0.05 (SAS V.9.1, SAS Institute, Cary, NC, USA).

3. Results

3.1. Accumulated Concentration of Amp in HLB-Affected Citrus after Nano-Formulation Treatment and the Effect on HLB

The droplet size of the Nano-Cre-Amp nano-formulation was significantly smaller than that of Nano-soy-Amp (16.27 ± 0.31 nm vs. 211.64 ± 24.86 nm; p = 0.0001) (Figure 1A). Four days after these nano-formulations were applied to HLB-affected citrus, citrus treated with the Nano-Cre-Amp formulation had the highest Amp concentration (56.06 ± 7.29 ng/g), which was substantially higher than that in plants treated with Nano-Soy-Amp (21.70 ± 6.20 ng/g) or Amp solution (9.03 ± 5.13 ng/g) (p = 0.0231) (Figure 1B). No Amp was detected in tissue from plants treated with tap water (CK). Taken together, the accumulated concentration of Amp inside the HLB-affected citrus treated with Nano-Cre-Amp, Nano-Soy-Amp, and Amp solution was high, medium, and low, respectively.
Consistent with the delivery of antimicrobial Amp into HLB-affected citrus by Nano-Cre-Amp (high), Nano-Soy-Amp (medium), and Amp solution (low) treatments, CLas titers were significantly reduced at 120 and 240 days after the initial treatment (Figure 2). No HLB symptoms were observed in any of the Amp-treated plants—regardless of delivery method—240 days after the initial treatment (Figure 3). The tap water treatment (CK) did not significantly suppress CLas titers in HLB-affected citrus (p = 0.0833) (Figure 2), and CK-treated plants continued to display HLB symptoms (Figure 3).

3.2. Transcriptome Profiles of HLB-Affected Citrus in Response to Amp at Different Concentrations

The transcriptome expression profiles in HLB-affected citrus in response to Amp were investigated by RNA-seq analysis. Between 56,612,324 and 59,582,280 reads per sample were obtained and 89.37% to 90.02%, respectively, of the reads uniquely mapped to the reference genome (Table S2). Totally, 2019 up-regulated and 2088 down-regulated DEGs were expressed in HLB-affected citrus treated with Nano-Cre-Amp (high), Nano-Soy-Amp (medium), and Amp solution (low) (Figure 4). The most up (1003)- and down (977)-regulated DEGs were found in HLB-affected citrus treated with Amp solution (low), followed by citrus treated with Nano-Cre-Amp (high) (320 up- and 290 down-regulated DEGs), and Nano-Soy-Amp (low) (237 up- and 277-down regulated DEGs) (Figure 4). In accordance with the number of up- and down-regulated DEGs, the Amp solution had the greatest impact on magnitude of the gene expression, followed by Nano-Cre-Amp (high) and Nano-Soy-Amp (medium). Meanwhile, 110 up- and 136 down-regulated DEGs were shared across all three treatments.
In the GO annotation, 379, 329, and 801 up-regulated DEGs were identified in Nano-Cre-Amp (high), Nano-Soy-Amp (medium), and Amp solution (low), respectively. In addition, 371, 412, and 803 down-regulated DEGs, respectively, were annotated in these three treatments. These DEGs were assigned to at least one term in the GO biological process, cellular component, and molecular function categories. In the cellular component categories, cell part, organelle, cell, and membrane were the most highly represented groups for both up-regulated and down-regulated DEGs (Tables S3 and S4). In molecular function categories, most up-regulated and down-regulated DEGs were enriched in binding and catalytic activity, whereas cellular process, single-organism process, and metabolic process dominated biological processes (Tables S3 and S4).
The KEGG pathway database was used to identify metabolic pathways in which Nano-Cre-Amp (high), Nano-Soy-Amp (medium), and Amp solution (low) were involved and enriched. In this study, KEGG analysis assigned the DEGs to 114 metabolic pathways. Most up-regulated DEGs were enriched in protein processing in the endoplasmic reticulum pathway (DEG proportion > 10%), followed by biosynthesis of amino acids, starch and sucrose metabolism, plant hormone signal transduction, amino sugar and nucleotide sugar metabolism, spliceosome, endocytosis, carbon metabolism, and plant-pathogen interaction (DEG proportion > 3%) (Figure 5). On the other hand, most down-regulated DEGs were enriched in plant hormone signal transduction, glycolysis/gluconeogenesis, phenylpropanoid biosynthesis, and carbon metabolism (DEG proportion > 3%) (Figure 6).

3.3. Verification of RNA-Seq Data with RT-qPCR

To confirm our transcriptomic data, 15 DEGs were selected for RT-qPCR. All 15 DEGs showed a change in transcript abundance in the RT-qPCR assay that was in accordance with that seen with RNA-seq. The results indicated that the change of transcript abundance of the 15 DEGs determined by RT-qPCR was the same as that detected by RNA-seq (Figure 7). The correlation coefficient (R2) between RT-qPCR and RNA-seq was >0.85 (Figure 7), indicating that the transcriptomic data were reliable.

3.4. DEGs Involved in Heat Shock Protein Up-Regulated by Ampicillin

In total, 20 DGEs involved in heat shock protein (Hsp) were up-regulated by all Amp treatments (Nano-Cre-Amp (high), Nano-Soy-Amp (medium), and Amp solution (low)) (Table S5). No down-regulated Hsp DEGs were seen with any of the Amp treatments (Table S6). Therefore, expression of Hsp DEGs appeared to be enhanced by ampicillin treatment. The 20 Hsp up-regulated DEGs were divided into five families including small Hsp (sHsp), Hsp40, Hsp70, Hsp90, and Hsp100. Another eight (Cs6g07320, Cs8g18360, Cs8g19540, orange1.1t05694, Cs8g19520, Cs5g18500, Cs9g14690, and Cs2g24360) and six (orange1.1t02795, Cs9g06800, Citrus_sinensis_newGene_1616, Cs8g18250, orange1.1t04990, and Citrus_sinensis_newGene_1609) DEGs belonged to the sHsp and Hsp70 family, respectively. The Hsp90 and Hsp100 family contained two (Cs9g19220 and Cs5g03150) and three DEGs (Cs5g11720, Citrus_sinensis_newGene_609 and Cs4g02760), respectively. Only one DEG (Cs2g16560) was seen for the Hsp40 family (Table 1).
The correlation between the Hsps and other DEGs was analyzed across the different Amp treatments (Nano-Cre-Amp (high), Nano-Soy-Amp (medium), and Amp-solution (low)) (Table S7). The DEGs that were significantly correlated with Hsps (1.00 > |R| > 0.99) were selected for further analysis. The results indicated that several DEGs involved in plant-pathogen interaction were closely related to Hsp gene expression. For instance, most DEGs (including L-ascorbate peroxidase 2, Transcription factor TCP19, TMV resistance protein N, Protein NTM1-like 8, WRKY transcription factor 19, and disease resistance protein) involved in plant defense were up-regulated by Amp treatment, whereas two DEGs, Polyubiquitin 10 (Citrus_sinensis_newGene_375) and Dehydration-responsive element-binding protein 1C (Cs5g12350), were down-regulated by Amp (Table S8). The Ferruginol synthase gene (orange1.1t04983) that is involved in secondary metabolism was up-regulated by Amp, and the other DEGs were down-regulated by Amp (Table S8). In the plant hormone pathway, Amp up-regulated expression of the Auxin-induced in root cultures protein 12 gene (Cs7g16820), and down-regulated other DEGs such as Gibberellic acid methyltransferase 2 (Cs9g03520). In RNA processing, the expression of Serine/arginine-rich SC35-like splicing factor SCL30A (Cs9g02960), Splicing factor U2af small subunit A (orange1.1t05219), Mediator of RNA polymerase II (Cs3g14220), Probable helicase MAGATAMA 3 (Cs3g02350), RNA-dependent RNA polymerase 6 (Cs7g05350), Putative pentatricopeptide repeat-containing protein (Cs8g08520), Glycine-rich RNA-binding protein (orange1.1t02557), and Double-stranded RNA-binding protein 1 (Cs3g17120) was enhanced by Amp (Table S8).

4. Discussion

Due to public concerns about the emergence of antibiotic-resistant bacteria and their potential effects on humans, use of Amp in citrus crops in commercial groves has not yet been approved by the Environmental Protection Agency (EPA) or other regulatory agencies. However, Amp is an effective chemical compound for combating CLas [19,23], and understanding the mechanism of Amp against CLas in HLB-affected citrus may be valuable for management of citrus HLB. In this study, HLB-affected citrus trees displayed no HLB symptoms and CLas titers were significantly reduced 240 days after treatment with different concentrations of Amp (Nano-Cre-Amp (High), Nano-Soy-Amp (Medium), and Amp solution (Low)) (Figure 2). Furthermore, 20 DEGs associated with heat shock proteins (Hsp) divided among five families including sHsp, Hsp40, Hsp70, Hsp90, and Hsp100, were up-regulated with all accumulated concentrations of Amp (Table 1), and this Amp-induced Hsp production could contribute to citrus defense against CLas.
As chaperones, Hsps promote folding, assembly, translocation, and degradation of proteins that are involved in many normal cellular processes. Hsps can also stabilize proteins and membranes as well as assist in protein refolding under heat stress conditions. However, since the discovery of Hsps/chaperones, these factors have been shown to play roles beyond heat stress management and are involved in responses to other stressors, including drought, salt, cold, heavy metals, oxidative stress, and chemicals stress [36,37]. Therefore, up-regulation of Hsps in HLB-affected citrus may be induced by Amp, which was considered to act as a Hsp stimulator (Table 1). Hsps may also play an important role in plant defense against pathogens. The functions of sHsp, Hsp40, Hsp70, and Hsp90 in plant pathogen defense are currently the most well-characterized. As a positive regulator of plant immunity, these three Hsp families are involved in resistance (R) proteins, reactive oxygen species (ROS) burst, cell death, and transcription factors that activate plant defense responses [38,39,40,41,42,43,44,45]. However, there are few studies about the function of Hsp100 in plant defense against pathogens [46]. Results of a recent study indicated that expression of 68 of 133 Hsps is significantly upregulated in HLB-tolerance citrus cultivars [47]. Moreover, several Hsps were also shown to be activated in HLB-affected citrus exposed to heat or treated with sulphonamide antibiotics, which can also mitigate HLB symptoms [18,48]. Therefore, up-regulation of Hsps may be related to tolerance to CLas, but the precise role of Hsps in citrus defense against CLas is still unknown.
In this study, several DEGs associated with R proteins, transcription factors, RNA metabolism, secondary metabolism, and plant hormones were significantly related to the up-regulated Hsps (Table S8), which may be related to citrus defense against CLas. The expression of two R protein genes TMV resistance protein N (orange1.1t04292) and disease resistance protein (Cs1g12160) was induced by Amp at all concentrations (Tables S7 and S8). TMV resistance protein N was negatively related to sHsp (Cs8g18360, Cs8g19520) and Hsp70 (Citrus_sinensis_newGene_1616, Cs9g06800), whereas disease resistance protein was negatively related to Hsp90 (Cs5g03150). A previous study indicated that several R proteins such as TMV resistance protein N were down-regulated by CLas infection [49]. This result suggests that CLas could secrete some effectors into the host cells and subvert host defense responses by affecting expression of some critical defense-related genes. As such, induction of R protein expression may confer resistance to citrus HLB.
Two transcription factor genes (WRKY19 (Cs5g24240)) and TCP19 (Cs9g16600) were up-regulated by Amp. WRKY19 and TCP19 were negatively (Cs5g18500 and Cs6g07320) and positively (Cs8g19540) related, respectively, to Hsps (Tables S7 and S8). CLas infection also affects the expression of genes encoding proteins containing the WRKY domain, including WRKY4, WRKY23, and WRKY30 [49,50]. Many WRKY domain-containing proteins are involved in plant defense responses. For instance, WRKY19 gene can regulate basal levels of Arabidopsis thaliana immunity to pathogen infection [51]. In addition, expression of Teosinte branched1/Cincinnata/proliferating cell factor (TCP) family members is down-regulated in response to CLas infection [52,53]. However, several studies indicated that multiple TCP factors (TCP13, TCP14, TCP15, TCP19, and TCP21) are targeted by effectors from bacterial pathogens Pseudomonas syringae (Psy). Moreover, plants lacking TCP13, TCP14, or TCP19 displayed enhanced disease susceptibility phenotypes [54,55]. Effectors of CLas may disturb expression of TCP genes such as TCP19 in citrus that is involved in defense against CLas. Therefore, WRKY transcription factor 19 and Transcription factor TCP19 may play a vital role in citrus immunity to CLas.
Alternative splicing (AS) describes the processing of a single pre-mRNA to produce multiple transcript isoforms. In recent, AS has been recognized as an important regulatory mechanism in plant defense against pathogen infections [56,57,58]. In this study, two splicing factor genes (serine/arginine-rich SC35-like splicing factor SCL30A (Cs9g02960) and splicing factor U2af small subunit A (orange1.1t05219)) were positively related to Hsps (Cs8g19540) and up-regulated by Amp (Tables S7 and S8). Glycine-rich RNA-binding protein gene (Cs4g02760) was negatively associated with Hsp70 (Cs4g02760), the Double-stranded RNA-binding protein 1 gene was positively related to sHsp (orange1.1t05694 and Cs9g14690). Hsp70 (orange1.1t04990) expression was also triggered by Amp (Table S8). Some splicing factors such as serine/arginine-rich splicing factor play a key role in plant defense and HR-like cell death [59,60]. RNA-binding proteins (RBPs) have been implicated at each level of RNA processing. More recent studies have characterized a number of plant RBPs and revealed their roles in plant immune responses [61,62]. In addition, expression of the RNA-dependent RNA polymerase 6 (Cs7g05350) gene, which was positively related to Hsp90 (Cs5g03150), was also enhanced by Amp (Tables S7 and S8). Several studies demonstrated that induction of RNA-dependent RNA polymerase activity plays a crucial role in plant defenses against pathogens [63,64]. Results of the present study showing up-regulated DEGs for splicing factor, RNA-binding protein, and RNA-dependent RNA polymerase, suggest that these genes may be involved in citrus immunity against CLas.
CLas-triggered ROS production is localized in the phloem-enriched bark tissue and is followed by systemic cell death of companion and sieve element cells. Foliar spray of gibberellin (GA) in HLB-affected citrus is known to have immunoregulatory activities [65], and antioxidants that reduce H2O2 concentrations and cell death in phloem tissues can mitigate HLB symptoms [66]. The L-ascorbate peroxidase 2 gene (Cs6g04140), which was negatively related to sHsp (Cs9g14690), was induced by Amp (Tables S7 and S8). Ascorbate peroxidase can rapidly scavenge H2O2 in the ascorbic acid and glutathione cycle [67]. Gibberellic acid methyltransferase 2 (Cs9g03520), which encodes a GA-inactivating enzyme, was positively related to sHsp (Cs6g07320) and Hsp100 (Cs4g02760) [68] and was down-regulated by Amp (Tables S7 and S8). Therefore, up-regulation of L-ascorbate peroxidase 2 and down-regulation of the Gibberellic acid methyltransferase 2 gene by Amp may be beneficial for alleviating HLB symptoms. Moreover, the ferruginol synthase gene (orange1.1t04983) was also induced by Amp and was positively associated with sHsp (orange1.1t05694 and Cs9g14690) and Hsp70 (orange1.1t04990) (Tables S7 and S8). Ferruginol synthase is involved in the biosynthesis of labdane-related diterpenes. In higher plants, diterpenes have many functions as hormones, antioxidants, antimicrobials, and defense-related molecules [69,70,71,72]. In this study, the up-regulated ferruginol synthase gene was also related to citrus defense against HLB.

5. Conclusions

Amp is typically considered to have antimicrobial activity against bacteria. However, in this study, Amp was shown to act as an Hsp stimulator that induced expression of many Hsps in HLB-affected citrus. These Hsps were related to several defense genes that express R proteins, transcription factors, splicing factors, RNA-binding proteins, RNA-dependent RNA polymerase, Gibberellic acid methyltransferase 2, L-ascorbate peroxidase 2, and ferruginol synthase, which together likely contributed to reductions in CLas titers and the mitigation of disease symptoms in HLB-affected citrus (Figure 8). In addition, the expression of defense genes was positively or negatively related to Hsps in response to treatment with Amp at different concentrations. These findings suggest that treatment with optimized concentrations of citrus inducers can be used to control citrus HLB. However, the exact mechanisms of interaction between the Hsps and defense genes remain unknown and require additional research. This study revealed that up-regulated Hsp genes and related defense genes may confer tolerance against CLas in citrus plants, and could provide a foundation for the development of novel strategies to combat HLB.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12061356/s1, Table S1. Primer sequences used to amplify fragments of DEGs. Table S2. Statistics for library sequencing. Nano-Cre-Amp, Nano-Soy-Amp, and Amp solutions are indicated as high, medium, and low Amp concentrations, respectively, in HLB-affected citrus. Table S3. Proportion (%) of up-regulated DEGs among total genes in the respective GO category following Amp treatment at different concentrations. Nano-Cre-Amp, Nano-Soy-Amp, and Amp solution are indicated as high, medium, and low Amp concentrations, respectively, in HLB-affected citrus. Table S4. Proportion (%) of down-regulated DEGs among total genes in the respective GO category under Amp treatment at different concentrations. Nano-Cre-Amp, Nano-Soy-Amp, and Amp solution are indicated as high, medium, and low Amp concentrations, respectively, in HLB-affected citrus. Table S5. Up-regulated DEGs present with all Amp concentrations. Nano-Cre-Amp, Nano-Soy-Amp, and Amp solution are indicated as high, medium, and low Amp concentrations, respectively, in HLB-affected citrus. Table S6. Down-regulated DEGs present with all Amp concentrations. Nano-Cre-Amp, Nano-Soy-Amp, and Amp solution are indicated as high, medium, and low Amp concentrations, respectively, in HLB-affected citrus. Table S7. Correlation analysis of Hsps and all DEGs. Table S8. Correlation analysis of Hsps and expression of genes related to pathogen defense.

Author Contributions

C.Y., Y.D. and M.Z. performed the experiments; C.Y., H.H. and X.L. analyzed the data; C.Y. and M.Z. contributed reagents/materials/analysis tools; G.F., C.P. and Y.D. wrote and revised the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This work is funded by Fujian Provincial Department of Science & Technology (Project 2020R10280017 and 2019R1028-13), Guangxi Provincial Department of Science & Technology (Guike AA 18118046), Fujian Academy of Agricultural Sciences (Project DC2017-5, A2017-1).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

We greatly appreciate Bioscience Editing Solutions for critically reading this paper and providing helpful suggestions.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Bové, J.M. Huanglongbing: A destructive, newly-emerging, century-old disease of citrus. J. Plant Pathol. 2006, 88, 7–37. [Google Scholar]
  2. Gottwald, T.R. Current epidemiological understanding of citrus huanglongbing. Annu. Rev. Phytopathol. 2010, 48, 119–139. [Google Scholar] [CrossRef] [Green Version]
  3. Jagoueix, S.; Bove, J.-M.; Garnier, M. The phloem-limited bacterium of greening disease of citrus is a member of the α subdivision of the Proteobacteria. Int. J. Syst. Evol. Microbiol. 1994, 44, 379–386. [Google Scholar] [CrossRef] [Green Version]
  4. Sutton, B.D. Detection and Identification of Citrus Huanglongbing (Greening) in Florida, USA. In Proceedings of the Second International Citrus Canker and Huanglongbing Research Workshop, Orlando, FL, USA, 7–11 November 2005; p. 59. [Google Scholar]
  5. Graham, J.; Gottwald, T.; Setamou, M. Status of huanglongbing (HLB) outbreaks in Florida, California and Texas. Trop. Plant Pathol. 2020, 45, 265–278. [Google Scholar] [CrossRef]
  6. Li, S.; Wu, F.; Duan, Y.; Singerman, A.; Guan, Z. Citrus greening: Management strategies and their economic impact. HortScience 2020, 55, 604–612. [Google Scholar] [CrossRef] [Green Version]
  7. Singerman, A.; Rogers, M.E. The economic challenges of dealing with citrus greening: The case of Florida. J. Integr. Pest Manag. 2020, 11, 3. [Google Scholar] [CrossRef] [Green Version]
  8. U.S. Department of Agriculture. Florida Citrus Fruit Size and Drop 2020–21 Report; U.S. Department of Agriculture: Washington, DC, USA, 2022.
  9. Wang, N.; Trivedi, P. Citrus huanglongbing: A newly relevant disease presents unprecedented challenges. Phytopathology 2013, 103, 652–665. [Google Scholar] [CrossRef] [Green Version]
  10. Blaustein, R.A.; Lorca, G.L.; Teplitski, M. Challenges for managing Candidatus Liberibacter spp. (Huanglongbing disease pathogen): Current control measures and future directions. Phytopathology 2018, 108, 424–435. [Google Scholar] [CrossRef] [Green Version]
  11. Ryals, J.A.; Neuenschwander, U.H.; Willits, M.G.; Molina, A.; Steiner, H.-Y.; Hunt, M.D. Systemic acquired resistance. Plant Cell 1996, 8, 1809. [Google Scholar] [CrossRef] [Green Version]
  12. Weller, D.M.; Mavrodi, D.V.; Van Pelt, J.A.; Pieterse, C.M.; Van Loon, L.C.; Bakker, P.A. Induced systemic resistance in Arabidopsis thaliana against Pseudomonas syringae pv. tomato by 2, 4-diacetylphloroglucinol-producing Pseudomonas fluorescens. Phytopathology 2012, 102, 403–412. [Google Scholar] [CrossRef] [Green Version]
  13. Walters, D.R.; Ratsep, J.; Havis, N.D. Controlling crop diseases using induced resistance: Challenges for the future. J. Exp. Bot. 2013, 64, 1263–1280. [Google Scholar] [CrossRef]
  14. Walters, D.; Walsh, D.; Newton, A.; Lyon, G. Induced resistance for plant disease control: Maximizing the efficacy of resistance elicitors. Phytopathology 2005, 95, 1368–1373. [Google Scholar] [CrossRef] [Green Version]
  15. Hu, J.; Jiang, J.; Wang, N. Control of citrus Huanglongbing via trunk injection of plant defense activators and antibiotics. Phytopathology 2018, 108, 186–195. [Google Scholar] [CrossRef] [Green Version]
  16. Li, J.; Trivedi, P.; Wang, N. Field evaluation of plant defense inducers for the control of citrus huanglongbing. Phytopathology 2016, 106, 37–46. [Google Scholar] [CrossRef] [Green Version]
  17. Canales, E.; Coll, Y.; Hernández, I.; Portieles, R.; Rodríguez García, M.; López, Y.; Aranguren, M.; Alonso, E.; Delgado, R.; Luis, M. ‘Candidatus Liberibacter asiaticus’, causal agent of citrus Huanglongbing, is reduced by treatment with Brassinosteroids. PLoS ONE 2016, 11, e0146223. [Google Scholar] [CrossRef]
  18. Yang, C.; Powell, C.A.; Duan, Y.; Ancona, V.; Huang, J.; Zhang, M. Transcriptomic analysis reveals root metabolic alteration and induction of huanglongbing resistance by sulphonamide antibiotics in huanglongbing-affected citrus plants. Plant Pathol. 2020, 69, 733–743. [Google Scholar] [CrossRef]
  19. Yang, C.; Powell, C.A.; Duan, Y.; Shatters, R.; Zhang, M. Antimicrobial nanoemulsion formulation with improved penetration of foliar spray through citrus leaf cuticles to control citrus huanglongbing. PLoS ONE 2015, 10, e0133826. [Google Scholar] [CrossRef] [Green Version]
  20. Zhang, M.; Guo, Y.; Powell, C.A.; Doud, M.S.; Yang, C.; Duan, Y. Effective antibiotics against ‘Candidatus Liberibacter asiaticus’ in HLB-affected citrus plants identified via the graft-based evaluation. PLoS ONE 2014, 9, e111032. [Google Scholar] [CrossRef] [Green Version]
  21. Spratt, B.G.; Cromie, K.D. Penicillin-binding proteins of gram-negative bacteria. Clin. Infect. Dis. 1988, 10, 699–711. [Google Scholar] [CrossRef]
  22. Ikenaga, T.; Kitamura, Y. Penicillin derivatives induce chemical structure-dependent root development, and application for plant transformation. Plant Cell Rep. 2004, 22, 668–677. [Google Scholar]
  23. Zhang, M.; Duan, Y.; Zhou, L.; Turechek, W.W.; Stover, E.; Powell, C.A. Screening molecules for control of citrus huanglongbing using an optimized regeneration system for ‘Candidatus Liberibacter asiaticus’-infected periwinkle (Catharanthus roseus) cuttings. Phytopathology 2010, 100, 239–245. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Yang, C.; Hu, H.; Wu, Y.; Lin, X.; Fan, G.; Duan, Y.; Powell, C.; Ancona, V.; Zhang, M. Metagenomic Analysis Reveals the Mechanism for the Observed Increase in Antibacterial Activity of Penicillin against Uncultured Bacteria Candidatus Liberibacter asiaticus Relative to Oxytetracycline in Planta. Antibiotics 2020, 9, 874. [Google Scholar] [CrossRef] [PubMed]
  25. Zhang, M.; Powell, C.A.; Benyon, L.S.; Zhou, H.; Duan, Y. Deciphering the bacterial microbiome of citrus plants in response to ‘Candidatus Liberibacter asiaticus’-infection and antibiotic treatments. PLoS ONE 2013, 8, e76331. [Google Scholar]
  26. Yang, C.; Powell, C.; Duan, Y.; Zhang, M. Characterization and antibacterial activity of oil-in-water Nano-emulsion formulation against Candidatus Liberibacter asiaticus. Plant Dis. 2016, 100, 2448–2454. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Li, W.; Hartung, J.S.; Levy, L. Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing. J. Microbiol. Methods 2006, 66, 104–115. [Google Scholar] [CrossRef]
  28. Xu, Q.; Chen, L.-L.; Ruan, X.; Chen, D.; Zhu, A.; Chen, C.; Bertrand, D.; Jiao, W.-B.; Hao, B.-H.; Lyon, M.P. The draft genome of sweet orange (Citrus sinensis). Nat. Genet. 2013, 45, 59–66. [Google Scholar] [CrossRef]
  29. Langmead, B.; Trapnell, C.; Pop, M.; Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009, 10, 1–10. [Google Scholar] [CrossRef] [Green Version]
  30. Mortazavi, A.; Williams, B.A.; McCue, K.; Schaeffer, L.; Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 2008, 5, 621–628. [Google Scholar] [CrossRef]
  31. Li, B.; Dewey, C.N. RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinform. 2011, 12, 1–16. [Google Scholar] [CrossRef] [Green Version]
  32. Conesa, A.; Götz, S.; García-Gómez, J.M.; Terol, J.; Talón, M.; Robles, M. Blast2GO: A universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 2005, 21, 3674–3676. [Google Scholar] [CrossRef] [Green Version]
  33. Koressaar, T.; Remm, M. Enhancements and modifications of primer design program Primer3. Bioinformatics 2007, 23, 1289–1291. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Yan, J.; Yuan, F.; Long, G.; Qin, L.; Deng, Z. Selection of reference genes for quantitative real-time RT-PCR analysis in citrus. Mol. Biol. Rep. 2012, 39, 1831–1838. [Google Scholar] [CrossRef] [PubMed]
  35. Yang, C.; Powell, C.A.; Duan, Y.; Shatters, R.; Fang, J.; Zhang, M. Deciphering the bacterial microbiome in huanglongbing-affected citrus treated with thermotherapy and sulfonamide antibiotics. PLoS ONE 2016, 11, e0155472. [Google Scholar] [CrossRef] [PubMed]
  36. Swindell, W.R.; Huebner, M.; Weber, A.P. Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways. BMC Genom. 2007, 8, 1–15. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Niu, Y.; Norouzitallab, P.; Baruah, K.; Dong, S.; Bossier, P. A plant-based heat shock protein inducing compound modulates host–pathogen interactions between Artemia franciscana and Vibrio campbellii. Aquaculture 2014, 430, 120–127. [Google Scholar] [CrossRef]
  38. Breiman, A. Plant Hsp90 and its co-chaperones. Curr. Protein Pept. Sci. 2014, 15, 232–244. [Google Scholar] [CrossRef]
  39. Xu, G.; Li, S.; Xie, K.; Zhang, Q.; Wang, Y.; Tang, Y.; Liu, D.; Hong, Y.; He, C.; Liu, Y. Plant ERD2-like proteins function as endoplasmic reticulum luminal protein receptors and participate in programmed cell death during innate immunity. Plant J. 2012, 72, 57–69. [Google Scholar] [CrossRef]
  40. Van Ooijen, G.; Lukasik, E.; Van Den Burg, H.A.; Vossen, J.H.; Cornelissen, B.J.; Takken, F.L. The small heat shock protein 20 RSI2 interacts with and is required for stability and function of tomato resistance protein I-2. Plant J. 2010, 63, 563–572. [Google Scholar] [CrossRef] [Green Version]
  41. Foley, R.C.; Gleason, C.A.; Anderson, J.P.; Hamann, T.; Singh, K.B. Genetic and genomic analysis of Rhizoctonia solani interactions with Arabidopsis; evidence of resistance mediated through NADPH oxidases. PLoS ONE 2013, 8, e56814. [Google Scholar] [CrossRef]
  42. Piterková, J.; Luhová, L.; Mieslerová, B.; Lebeda, A.; Petřivalský, M. Nitric oxide and reactive oxygen species regulate the accumulation of heat shock proteins in tomato leaves in response to heat shock and pathogen infection. Plant Sci. 2013, 207, 57–65. [Google Scholar] [CrossRef]
  43. Yogendra, K.N.; Kumar, A.; Sarkar, K.; Li, Y.; Pushpa, D.; Mosa, K.A.; Duggavathi, R.; Kushalappa, A.C. Transcription factor StWRKY1 regulates phenylpropanoid metabolites conferring late blight resistance in potato. J. Exp. Bot. 2015, 66, 7377–7389. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Ito, M.; Ohnishi, K.; Hikichi, Y.; Kiba, A. Molecular chaperons and co-chaperons, Hsp90, RAR1, and SGT1 negatively regulate bacterial wilt disease caused by Ralstonia solanacearum in Nicotiana benthamiana. Plant Signal. Behav. 2015, 10, e970410. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Liu, J.Z.; Whitham, S.A. Overexpression of a soybean nuclear localized type–III DnaJ domain-containing HSP40 reveals its roles in cell death and disease resistance. Plant J. 2013, 74, 110–121. [Google Scholar] [CrossRef]
  46. Enikeev, A.; Kopytina, T.; Semenova, L.; Shafikova, T.; Gamanets, L.; Volkova, O.; Shvetsov, S.; Rusaleva, T. Tobacco cell cultures transformed by the hsp 101 gene exhibit an increased resistance to potassium fluoride. In Proceedings of the Doklady Biological Sciences; Springer Nature BV: Dordrecht, The Netherlands, 2010; p. 29. [Google Scholar]
  47. Wu, B.; Yu, Q.; Deng, Z.; Duan, Y.; Luo, F.; Gmitter, F. A chromosome-level phased Citrus sinensis genome facilitates understanding Huanglongbing tolerance mechanisms at the allelic level in an irradiation induced mutant. bioRxiv 2022. [Google Scholar] [CrossRef]
  48. Nwugo, C.C.; Doud, M.S.; Duan, Y.-P.; Lin, H. Proteomics analysis reveals novel host molecular mechanisms associated with thermotherapy of ‘Ca. Liberibacter asiaticus’-infected citrus plants. BMC Plant Biol. 2016, 16, 1–15. [Google Scholar] [CrossRef] [Green Version]
  49. Aritua, V.; Achor, D.; Gmitter, F.G.; Albrigo, G.; Wang, N. Transcriptional and microscopic analyses of citrus stem and root responses to Candidatus Liberibacter asiaticus infection. PLoS ONE 2013, 8, e73742. [Google Scholar] [CrossRef] [Green Version]
  50. Mafra, V.; Martins, P.K.; Francisco, C.S.; Ribeiro-Alves, M.; Freitas-Astúa, J.; Machado, M.A. Candidatus Liberibacter americanus induces significant reprogramming of the transcriptome of the susceptible citrus genotype. BMC Genom. 2013, 14, 1–15. [Google Scholar] [CrossRef] [Green Version]
  51. Warmerdam, S.; Sterken, M.G.; Sukarta, O.C.; Van Schaik, C.C.; Oortwijn, M.E.; Lozano-Torres, J.L.; Bakker, J.; Smant, G.; Goverse, A. The TIR-NB-LRR pair DSC1 and WRKY19 contributes to basal immunity of Arabidopsis to the root-knot nematode Meloidogyne incognita. BMC Plant Biol. 2020, 20, 1–14. [Google Scholar] [CrossRef]
  52. Zhong, Y.; Cheng, C.-Z.; Jiang, N.-H.; Jiang, B.; Zhang, Y.-Y.; Wu, B.; Hu, M.-L.; Zeng, J.-W.; Yan, H.-X.; Yi, G.-J. Comparative transcriptome and iTRAQ proteome analyses of citrus root responses to Candidatus Liberibacter asiaticus infection. PLoS ONE 2015, 10, e0126973. [Google Scholar] [CrossRef] [Green Version]
  53. Xu, M.; Li, Y.; Zheng, Z.; Dai, Z.; Tao, Y.; Deng, X. Transcriptional analyses of mandarins seriously infected by ‘Candidatus Liberibacter asiaticus’. PLoS ONE 2015, 10, e0133652. [Google Scholar] [CrossRef]
  54. Mukhtar, M.S.; Carvunis, A.-R.; Dreze, M.; Epple, P.; Steinbrenner, J.; Moore, J.; Tasan, M.; Galli, M.; Hao, T.; Nishimura, M.T. Independently evolved virulence effectors converge onto hubs in a plant immune system network. Science 2011, 333, 596–601. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Weßling, R.; Epple, P.; Altmann, S.; He, Y.; Yang, L.; Henz, S.R.; McDonald, N.; Wiley, K.; Bader, K.C.; Gläßer, C. Convergent targeting of a common host protein-network by pathogen effectors from three kingdoms of life. Cell Host Microbe 2014, 16, 364–375. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Zhang, X.-C.; Gassmann, W. Alternative splicing and mRNA levels of the disease resistance gene RPS4 are induced during defense responses. Plant Physiol. 2007, 145, 1577–1587. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  57. Dinesh-Kumar, S.; Baker, B.J. Alternatively spliced N resistance gene transcripts: Their possible role in tobacco mosaic virus resistance. Proc. Natl. Acad. Sci. USA 2000, 97, 1908–1913. [Google Scholar] [CrossRef] [Green Version]
  58. Zhang, X.-C.; Gassmann, W. RPS4-mediated disease resistance requires the combined presence of RPS4 transcripts with full-length and truncated open reading frames. Plant Cell 2003, 15, 2333–2342. [Google Scholar] [CrossRef]
  59. Kumar, K.R.R.; Kirti, P. Novel role for a serine/arginine-rich splicing factor, AdRSZ21 in plant defense and HR-like cell death. Plant Mol. Biol. 2012, 80, 461–476. [Google Scholar] [CrossRef]
  60. Chen, X.; Hao, L.; Pan, J.; Zheng, X.; Jiang, G.; Jin, Y.; Gu, Z.; Qian, Q.; Zhai, W.; Ma, B. SPL5, a cell death and defense-related gene, encodes a putative splicing factor 3b subunit 3 (SF3b3) in rice. Mol. Breed. 2012, 30, 939–949. [Google Scholar] [CrossRef]
  61. Fu, Z.Q.; Guo, M.; Jeong, B.-R.; Tian, F.; Elthon, T.E.; Cerny, R.L.; Staiger, D.; Alfano, J.R. A type III effector ADP-ribosylates RNA-binding proteins and quells plant immunity. Nature 2007, 447, 284–288. [Google Scholar] [CrossRef]
  62. He, B.; Cai, Q.; Qiao, L.; Huang, C.-Y.; Wang, S.; Miao, W.; Ha, T.; Wang, Y.; Jin, H. RNA-binding proteins contribute to small RNA loading in plant extracellular vesicles. Nat. Plants 2021, 7, 342–352. [Google Scholar] [CrossRef]
  63. Xie, Z.; Fan, B.; Chen, C.; Chen, Z. An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense. Proc. Natl. Acad. Sci. USA 2001, 98, 6516–6521. [Google Scholar] [CrossRef] [Green Version]
  64. Wagh, S.; Alam, M.; Kobayashi, K.; Yaeno, T.; Yamaoka, N.; Toriba, T.; Hirano, H.-Y.; Nishiguchi, M. Analysis of rice RNA-dependent RNA polymerase 6 (OsRDR6) gene in response to viral, bacterial and fungal pathogens. J. Gen. Plant Pathol. 2016, 82, 12–17. [Google Scholar] [CrossRef]
  65. Huot, B.; Yao, J.; Montgomery, B.L.; He, S.Y. Growth–defense tradeoffs in plants: A balancing act to optimize fitness. Mol. Plant 2014, 7, 1267–1287. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  66. Ma, W.; Pang, Z.; Huang, X.; Xu, J.; Pandey, S.S.; Li, J.; Achor, D.S.; Vasconcelos, F.N.; Hendrich, C.; Huang, Y. Citrus Huanglongbing is a pathogen-triggered immune disease that can be mitigated with antioxidants and gibberellin. Nat. Commun. 2022, 13, 1–13. [Google Scholar] [CrossRef] [PubMed]
  67. Shigeoka, S.; Ishikawa, T.; Tamoi, M.; Miyagawa, Y.; Takeda, T.; Yabuta, Y.; Yoshimura, K. Regulation and function of ascorbate peroxidase isoenzymes. J. Exp. Bot. 2002, 53, 1305–1319. [Google Scholar] [CrossRef]
  68. Varbanova, M.; Yamaguchi, S.; Yang, Y.; McKelvey, K.; Hanada, A.; Borochov, R.; Yu, F.; Jikumaru, Y.; Ross, J.; Cortes, D. Methylation of gibberellins by Arabidopsis GAMT1 and GAMT2. Plant Cell 2007, 19, 32–45. [Google Scholar] [CrossRef] [Green Version]
  69. Seo, S.; Gomi, K.; Kaku, H.; Abe, H.; Seto, H.; Nakatsu, S.; Neya, M.; Kobayashi, M.; Nakaho, K.; Ichinose, Y. Identification of natural diterpenes that inhibit bacterial wilt disease in tobacco, tomato and Arabidopsis. Plant Cell Physiol. 2012, 53, 1432–1444. [Google Scholar] [CrossRef] [Green Version]
  70. Bailey, J.; Carter, G.; Burden, R.; Wain, R. Control of rust diseases by diterpenes from Nicotiana glutinosa. Nature 1975, 255, 328–329. [Google Scholar] [CrossRef]
  71. Jasiński, M.; Stukkens, Y.; Degand, H.; Purnelle, B.; Marchand-Brynaert, J.; Boutry, M. A plant plasma membrane ATP binding cassette–type transporter is involved in antifungal terpenoid secretion. Plant Cell 2001, 13, 1095–1107. [Google Scholar] [CrossRef]
  72. Campbell, E.J.; Schenk, P.M.; Kazan, K.; Penninckx, I.A.; Anderson, J.P.; Maclean, D.J.; Cammue, B.P.; Ebert, P.R.; Manners, J.M. Pathogen-responsive expression of a putative ATP-binding cassette transporter gene conferring resistance to the diterpenoid sclareol is regulated by multiple defense signaling pathways in Arabidopsis. Plant Physiol. 2003, 133, 1272–1284. [Google Scholar] [CrossRef] [Green Version]
Agronomy 12 01356 g001 550
Figure 1. (A): Droplet size of Nano-Cre-Amp and Nano-Soy-Amp nano-formulations. (B): Accumulated concentration of Amp in HLB-affected citrus treated with Nano-Cre-Amp, Nano-Soy-Amp, or Amp solution. Different letters represent significant differences at the level of 0.05 (Duncan’s multiple range tests) (p < 0.05). Error bars indicate standard deviation. Six replicates in each treatment.
Figure 1. (A): Droplet size of Nano-Cre-Amp and Nano-Soy-Amp nano-formulations. (B): Accumulated concentration of Amp in HLB-affected citrus treated with Nano-Cre-Amp, Nano-Soy-Amp, or Amp solution. Different letters represent significant differences at the level of 0.05 (Duncan’s multiple range tests) (p < 0.05). Error bars indicate standard deviation. Six replicates in each treatment.
Agronomy 12 01356 g001
Agronomy 12 01356 g002 550
Figure 2. Clas titer in HLB-affected citrus in response to different concentration of Amp (Nano-Cre-Amp (high), Nano-Soy-Amp (medium), Amp solution (low), and tap water (CK)) Different letters represent significant differences at the level of 0.05 (Duncan’s multiple range tests) (p < 0.05). Error bars indicate standard deviation. Six replicates in each treatment.
Figure 2. Clas titer in HLB-affected citrus in response to different concentration of Amp (Nano-Cre-Amp (high), Nano-Soy-Amp (medium), Amp solution (low), and tap water (CK)) Different letters represent significant differences at the level of 0.05 (Duncan’s multiple range tests) (p < 0.05). Error bars indicate standard deviation. Six replicates in each treatment.
Agronomy 12 01356 g002
Agronomy 12 01356 g003 550
Figure 3. The symptom in HLB-affected citrus in response to different concentrations of Amp at 240 days after initial treatment. (A): Nano-Cre-Amp (high), (B): Nano-Soy-Amp (medium), (C): Amp solution (low), and (D): Tap water (CK).
Figure 3. The symptom in HLB-affected citrus in response to different concentrations of Amp at 240 days after initial treatment. (A): Nano-Cre-Amp (high), (B): Nano-Soy-Amp (medium), (C): Amp solution (low), and (D): Tap water (CK).
Agronomy 12 01356 g003
Agronomy 12 01356 g004 550
Figure 4. Number of up-regulated (A) and down-regulated (B) DEGs in HLB-affected citrus in response to treatment with different concentrations of Amp (Nano-Cre-Amp (high), Nano-Soy-Amp (medium), and Amp solution (low)).
Figure 4. Number of up-regulated (A) and down-regulated (B) DEGs in HLB-affected citrus in response to treatment with different concentrations of Amp (Nano-Cre-Amp (high), Nano-Soy-Amp (medium), and Amp solution (low)).
Agronomy 12 01356 g004
Agronomy 12 01356 g005 550
Figure 5. Proportion (%) of up-regulated DEGs among total genes in the respective KEGG pathway at different Amp concentration.
Figure 5. Proportion (%) of up-regulated DEGs among total genes in the respective KEGG pathway at different Amp concentration.
Agronomy 12 01356 g005
Agronomy 12 01356 g006 550
Figure 6. Proportion (%) of down-regulated DEGs among total genes in the respective KEGG pathway at different Amp concentration.
Figure 6. Proportion (%) of down-regulated DEGs among total genes in the respective KEGG pathway at different Amp concentration.
Agronomy 12 01356 g006
Agronomy 12 01356 g007 550
Figure 7. RT-qPCR validation of DEGs. Error bars indicate standard deviation. RT-qPCR was conducted with three biological repetitions.
Figure 7. RT-qPCR validation of DEGs. Error bars indicate standard deviation. RT-qPCR was conducted with three biological repetitions.
Agronomy 12 01356 g007
Agronomy 12 01356 g008 550
Figure 8. Overview of the relationship between Hsps and defense genes in HLB-affected citrus in response to Amp treatment.
Figure 8. Overview of the relationship between Hsps and defense genes in HLB-affected citrus in response to Amp treatment.
Agronomy 12 01356 g008
Table
Table 1. Family, expression, and annotation of DEGs involved in Hsp in HLB-affected citrus in response to treatment with the antibiotic ampicillin.
Table 1. Family, expression, and annotation of DEGs involved in Hsp in HLB-affected citrus in response to treatment with the antibiotic ampicillin.
Hsp FamiliesGene IDFold Change (Log2)Gene Annotation
Nano-Cre-AmpNano-Soy-AmpAmp Solution
sHspCs6g073203.012.892.5717.3 kDa class I heat shock protein
Cs8g183603.252.942.8617.4 kDa class I heat shock protein
Cs8g195402.933.782.7217.5 kDa class I heat shock protein
orange1.1t056941.872.101.1817.6 kDa class I heat shock protein
Cs8g195202.091.591.4618.5 kDa class I heat shock protein
Cs5g185004.494.213.6022.0 kDa heat shock protein
Cs9g146902.092.191.6723.5 kDa heat shock protein
Cs2g243601.882.442.75Small heat shock protein
Hsp40Cs2g165601.301.921.83Chaperone protein dnaJ
Hsp70orange1.1t027952.922.452.64Heat shock 70 kDa protein
Cs9g068002.221.821.69Heat shock 70 kDa protein
Citrus_sinensis_newGene_16162.011.661.58Heat shock 70 kDa protein
Cs8g182501.733.892.06Heat shock cognate 70 kDa protein
orange1.1t049902.682.752.54Heat shock cognate 70 kDa protein
Citrus_sinensis_newGene_16093.413.623.49Heat shock cognate 70 kDa protein
Hsp90Cs9g192201.721.311.67Heat shock protein 83
Cs5g031502.381.441.28Heat shock protein 83
Hsp100Cs5g117201.751.431.49Chaperone protein ClpB1
Citrus_sinensis_newGene_6092.421.532.11Chaperone protein ClpB1
Cs4g027601.751.671.02Chaperone protein ClpB1
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Yang, C.; Powell, C.; Duan, Y.; Lin, X.; Fan, G.; Hu, H.; Zhang, M. Key Role of Heat Shock Protein Expression Induced by Ampicillin in Citrus Defense against Huanglongbing: A Transcriptomics Study. Agronomy 2022, 12, 1356. https://doi.org/10.3390/agronomy12061356

AMA Style

Yang C, Powell C, Duan Y, Lin X, Fan G, Hu H, Zhang M. Key Role of Heat Shock Protein Expression Induced by Ampicillin in Citrus Defense against Huanglongbing: A Transcriptomics Study. Agronomy. 2022; 12(6):1356. https://doi.org/10.3390/agronomy12061356

Chicago/Turabian Style

Yang, Chuanyu, Charles Powell, Yongping Duan, Xiongjie Lin, Goucheng Fan, Hanqing Hu, and Muqing Zhang. 2022. "Key Role of Heat Shock Protein Expression Induced by Ampicillin in Citrus Defense against Huanglongbing: A Transcriptomics Study" Agronomy 12, no. 6: 1356. https://doi.org/10.3390/agronomy12061356

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop