Abstract

   Sugarcane white leaf (SCWL) disease, caused by Candidatus Phytoplasma
   sacchari, results in the most damage to sugarcane plantations. Some
   SCWL canes can grow unnoticed through the maturation phase,
   subsequently resulting in an overall low sugar yield, or they can be
   used accidentally as seed canes. In this work, 12-month-old SCWL and
   asymptomatic canes growing in the same field were investigated. An
   abundance of phytoplasma in SCWL canes affected growth and sugar
   content as well as alterations of transcriptomic profiles corresponding
   to several pathways that responded to the infection. Suppression of
   photosynthesis, porphyrin and chlorophyll metabolism, coupled with an
   increase in the expression of chlorophyllase, contributed to the
   reduction in chlorophyll levels and photosynthesis. Blockage of sucrose
   transport plausibly occurred due to the expression of sugar
   transporters in leaves but suppression in stalks, resulting in low
   sugar content in canes. Increased expression of genes associated with
   MAPK cascades, plant hormone signaling transduction, callose plug
   formation, the phenylpropanoid pathway, and calcium cascades positively
   promoted defense mechanisms against phytoplasma colonization by an
   accumulation of lignin and calcium in response to plant immunity.
   Significant downregulation of CPK plausibly results in a reduction in
   antioxidant enzymes and likely facilitates pathogen invasion, while
   expression of sesquiterpene biosynthesis possibly attracts the insect
   vectors for transmission, thereby enabling the spread of phytoplasma.
   Moreover, downregulation of flavonoid biosynthesis potentially
   intensifies the symptoms of SCWL upon challenge by phytoplasma. These
   SCWL sugarcane transcriptomic profiles describe the first comprehensive
   sugarcane–phytoplasma interaction during the harvesting stage.
   Understanding molecular mechanisms will allow for sustainable
   management and the prevention of SCWL disease—a crucial benefit to the
   sugar industry.

   Keywords: sugarcane white leaf, phytoplasma, transcriptome, plant
   defense response, biotic stress

1. Introduction

   Sugarcane (Saccharum sp.), a grass monocot plant in the Family Poaceae,
   is one of the most important economic crops for the sugar industry as
   well as the production of ethanol, molasses, and animal feed. Thailand
   is the second-largest sugar exporter after Brazil and is ranked fourth
   for sugar production worldwide (US Department of Agriculture,
   2023/2024). However, sugar production from cane has been seriously
   affected by several sugarcane diseases. One of the most destructive
   effects on sugar yield from canes in Thailand and other regions is
   sugarcane white leaf (SCWL) disease. The economic loss caused by SCWL
   disease on income from sugarcane cultivation was estimated to be around
   USD 30–40 million annually [[28]1].

   SCWL disease is caused by an obligate pathogenic bacterium that lacks a
   cell wall, Candidatus Phytoplasma sacchari, which so far has been
   impracticable to culture in laboratory conditions [[29]2]. SCWL
   phytoplasma is transmitted via insect vectors, Matsumuratettix
   hiroglyphicus Matsumura [[30]3] and Yamatotettix flavovittatus
   Matsumura [[31]4]. After transmission, the bacterium generally
   localizes in phloem and uses metabolic pathways from the sugarcane host
   [[32]5]. Based on 16S rRNA classification, SCWL phytoplasma is closely
   related to sugarcane grassy shoot (SCGS) and rice yellow dwarf (RYD)
   [[33]6,[34]7].

   Symptoms of SCWL disease can be observed in every growth stage of
   sugarcane from seed canes or cane setts to the mature stage. In
   general, new emerging buds and shoots from the cane setts emerge with
   bushy slim white leaves of a soft texture that grow from slender
   chlorotic shoots [[35]7]. Degrees of leaf chlorosis start from pale
   green to white with considerable tiller proliferation and stunting
   [[36]8]. Variation in symptoms depends on the stage of sugarcane growth
   and development of disease. Although SCWL sugarcane is recognized and
   eliminated while it is young, less symptomatic canes can be missed
   visually and remain in the field as SCWL carriers. At the maturation
   phase (12 months old), when sugarcanes contain the highest sugar yield
   [[37]9], such SCWL canes are harvested together with the healthy ones
   and result in a low yield of sugar production. Moreover, they could be
   unintentionally prepared as seed canes for the next propagation cycle
   that will quickly spread SCWL disease to other sugarcane plantations.

   In Thailand, Khon Kaen 3 (KK3) is one of the most popular sugarcane
   cultivars for the sugar industry due to its high sugar productivity and
   vigorous growth in various soil conditions [[38]10], but KK3 is not
   tolerant to SCWL disease. In this study, 12-month-old SCWL KK3, growing
   together with asymptomatic KK3 in the same field, were harvested and
   investigated. To determine the sugarcane genes that play important
   roles in SCWL pathogenesis, transcriptomic profiles of SCWL sugarcane
   leaves and stalks were undertaken as both organs perform the distinct
   metabolic functions of photosynthesis and sugar storage, respectively.
   Molecular mechanisms and related pathways of the sugarcane host
   affected by SCWL phytoplasma have been identified.

2. Results

2.1. Growth Characteristics and Phytoplasma Detection of SCWL Sugarcanes

   Twelve-month-old SCWL sugarcanes showed symptoms of chlorosis leaf,
   0.65 times smaller stalk diameter, 0.65 times shorter height, and 0.69
   times lower sugar content than those of asymptomatic sugarcanes
   collected from the same plantation ([39]Table 1, [40]Figure S1A–C). To
   evaluate the numbers of active phytoplasma in leaves and stalks of
   sugarcane samples, the gene expression ratio of 16S/18S rRNA of
   phytoplasma/sugarcane was calculated. SCWL sugarcanes harbored 10^4
   times more phytoplasma than those of asymptomatic canes ([41]Table 1).
   After propagation of the SCWL seed canes under greenhouse conditions
   for 2 months, newly emerged buds produced tillers and stunting with
   slim and narrow white leaves ([42]Figure S1D).

Table 1.

   Growth parameters, sugar content, and phytoplasma ratio of asymptomatic
   and symptomatic sugarcanes.
   Sugarcane Height (cm) Diameter (cm) Sugar Content (°Brix) 16S/18S rRNA
   Gene Ratio
   Leaves Stalks
   Asymptom 302.20 ± 12.77 3.34 ± 0.13 22.84 ± 0.25 2.35 × 10^−5 ± 0.82 ×
   10^−5 8.96 × 10^−5 ± 3.00 × 10^−5
   Symptom 199.20 ± 16.76 * 2.18 ± 0.16 * 15.86 ± 1.14 * 0.33 ± 0.10 *
   0.57 ± 0.14 *
   [43]Open in a new tab

   Asterisk (*) indicates significant differences by unpaired t-test
   analysis.

2.2. RNA-Sequencing and Assembly

   Total RNA-seq data of leaves and stalks of SCWL and asymptomatic
   sugarcanes were analyzed in triplicate (BioProject number PRJNA719388).
   More than 671 million raw reads (sequencing depth of 53.8–59.8 million
   of 150 bp paired-end read per library) were generated. About 647
   million clean reads were filtered and 69.48% to 80.66% of the reads
   were mapped to the monoploid sugarcane genome reference
   ([44]https://sugarcane-genome.cirad.fr; accessed on 14 February 2020)
   [[45]11] ([46]Table S1). Principal component analysis (PCA) of gene
   expression showed that SCWL and asymptomatic sugarcanes clustered
   separately in both leaves and stalks ([47]Figure 1) and the volcano
   plot showed a reasonable distribution of gene expression ([48]Figure
   S2). In leaves, 4799 and 1485 differentially expressed genes (DEGs;
   p-value < 0.05, log[2] FC > 1) were upregulated and downregulated,
   respectively, while in stalks, 994 and 743 DEGs were upregulated and
   downregulated, respectively ([49]Table 2). Co-expression of DEGs
   between leaves and stalks contained 330 upregulated and 92
   downregulated DEGs, respectively ([50]Table 2).

Figure 1.

   [51]Figure 1
   [52]Open in a new tab

   Principal component analysis (PCA) of gene expression from RNA-seq data
   of asymptomatic and symptomatic sugarcanes. AS, asymptomatic stalks;
   SS, symptomatic stalks; AL: asymptomatic leaves; SL, symptomatic
   leaves. The two principal components determine 93% of the total
   variance.

Table 2.

   Number of DEGs in gene ontology (GO) and KEGG pathway in SCWL leaves
   and stalks.
                     Category                         Number of DEGs
                                                    Leaves        Stalks
   Significantly expressed gene (p-value < 0.05)     6612          2173
   |log[2]Fold Change| > 1.0                         6284          1737
   - Upregulated genes                               4799          994
   - Downregulate genes                              1485          743
   Gene ontology                                 3711 (59.06%) 987 (56.82%)
   - Molecular function                              3350          901
   - Cellular component                               884          186
   - Biological process                              2523          675
   KEGG pathway                                  2248 (35.79%) 531 (30.57%)
   - Upregulated DEGs                                1701          294
   - Downregulated DEGs                               547          237
   [53]Open in a new tab

2.3. Gene Ontology (GO) Annotation and KEGG Pathway-Enrichment Analysis

   The significant upregulated and downregulated DEGs of leaves and stalks
   were annotated with GO terms ([54]Table 2). In leaves, DEGs were
   categorized and enriched in biological processes (e.g., photosynthesis,
   carbohydrate metabolic processes, generation of precursor metabolites
   and energy, cellular amino acid and protein metabolic processes),
   cellular component (membrane and thylakoid), and molecular function
   (e.g., transporter activity, catalytic activity, kinase/transferase
   activity, carbohydrate binding, and transcription factor activity)
   ([55]Figure S3A), while in stalks, significant DEGs were categorized in
   biological processes (e.g., regulation of metabolic process,
   macromolecule modification, regulation of gene expression, protein
   modification processes, and regulation of biological processes) and
   molecular function (e.g., transcription factor activity, transferase
   activity, kinase activity, and transporter activity) ([56]Figure S3B).

   As a result of the KEGG analysis, upregulated and downregulated DEGs of
   leaves and stalks were annotated for functionality ([57]Table 2). The
   most frequent pathways were metabolism, genetic information processing,
   environmental information processing, cellular processes, and
   organismal systems ([58]Figure 2). To attain pathway enrichment, the
   ‘piano in R’ package was used. The enriched DEGs in leaves were
   associated with various functions including photosynthesis, signal
   transduction, flavonoid biosynthesis, metabolism of terpenoids and
   polyketides ([59]Figure 3, [60]Table S2). The most enriched upregulated
   pathways included mitogen-activated protein kinase (MAPK) signaling
   pathways, monoterpenoid biosynthesis, and plant–pathogen interactions,
   while the most enriched downregulated pathways were photosynthesis,
   photosynthesis antenna proteins, porphyrin and chlorophyll metabolism,
   signal transduction, carotenoid biosynthesis, and carbon fixation in
   photosynthetic organisms ([61]Table S2). In stalks, the enriched DEGs
   included plant–pathogen interactions and protein processing in the
   endoplasmic reticulum pathway ([62]Figure 3, [63]Table S3). The most
   enriched upregulated pathways encompassed brassinosteroid biosynthesis,
   sesquiterpenoid and triterpenoid biosynthesis, while the most enriched
   downregulated pathways were starch and sucrose metabolism,
   glycolysis/gluconeogenesis, diterpenoid biosynthesis, flavonoid
   biosynthesis and carbon fixation in photosynthetic organisms ([64]Table
   S3).

Figure 2.

   [65]Figure 2
   [66]Open in a new tab

   Number of DEGs in each category of KEGG pathways in SCWL leaves and
   stalks.

Figure 3.

   [67]Figure 3
   [68]Open in a new tab

   Top 20 KEGG pathway enrichment of DEGs between SCWL and asymptomatic
   sugarcanes in leaves (A) and stalks (B). The rich factor is a ratio of
   the number of DEGs and the total genes. The size and color of the
   bubble represent the number of DEGs and the p-value, respectively.

2.4. Phytoplasma-Affected Metabolic Processes of Sugarcane

   From GO and KEGG pathway analyses, infection by phytoplasma evidently
   influenced the expression of many genes related to metabolic pathways
   in leaves and stalks including chlorophyll metabolism and
   photosynthesis, sucrose accumulation, plant–pathogen interactions,
   plant hormone signaling transduction, and secondary metabolites.

2.4.1. Chlorophyll Metabolism and Photosynthesis

   In SCWL leaves, phytoplasma significantly suppressed the photosynthetic
   pathway and chlorophyll content, resulting in an alteration of the
   downstream pathways. Most DEGs of porphyrin and chlorophyll metabolism
   were downregulated including protoporphyrin IX-related genes;
   chlorophyllide b reductase (NYC1, K13606), geranylgeranyl diphosphate
   (chlP, K10960), 7-hydroxymethyl chlorophyll a reductase (HCAR, K18010),
   magnesium chelatases (chlH, K03404; K03405), magnesium-protoporphyrin
   IX monomethyl ester cyclase (chlE, K04035), protochlorophyllide
   reductase (por, [69]K00218); and heme-related gene clusters [hemA
   ([70]K02492), hemC ([71]K01749), hemE ([72]K01599), hemL ([73]K01845),
   and hemY ([74]K00231)]; while chlorophyllase (K08099) and cytochrome c
   oxidase assembly protein subunit 15 (COX15, [75]K02259) were
   upregulated ([76]Figure 4 and [77]Table S4).

Figure 4.

   [78]Figure 4
   [79]Open in a new tab

   Effect of phytoplasma on photosynthesis and chlorophyll metabolism in
   SCWL sugarcane. Heatmaps of upregulated and downregulated DEGs of
   porphyrin and chlorophyll metabolism, photosynthesis, and
   photosynthesis antenna proteins are shown. Blue arrow, upregulation;
   red arrow, downregulation.

   Meanwhile, most DEGs of photosynthesis were downregulated including
   cytochrome b6/f complex subunits [PetA, [80]K02634; PetC, PetD,
   ([81]K02636–7)], photosystem I subunits (PsaE, [82]K02693; PsaH,
   [83]K02695; PsaK, [84]K02698; PsaN, [85]K02701), photosystem II Psb
   proteins [PsbC, [86]K02705; PsbP, [87]K02717; PsbS, [88]K03542; PsbW,
   [89]K02721; PsbY, PsbZ, ([90]K02723–4)], and photosynthetic electron
   transport, i.e., plastocyanin and ferredoxin [PetE, PetF
   ([91]K02638–9)]. Moreover, the DEGs of photosynthesis antenna protein
   were also downregulated including the light-harvesting chlorophyll
   protein complex [Lhca1, Lhca2, Lhca3, (K08907–9); Lhca5, Lhcb1, Lhcb2,
   Lhcb3, Lhcb4 (K08911–5) ([92]Figure 4 and [93]Table S4).

2.4.2. Regulation of Genes Related to Sucrose Accumulation

   The accumulation of sucrose was negatively affected in sugarcanes
   infected by phytoplasma. In leaves, transcripts related to sucrose
   accumulation were altered by most DEGs of carbon fixation in
   photosynthetic organisms including the upregulation of aspartate
   aminotransferase (GOT1, K14454), phosphoenolpyruvate carboxykinase
   (pecK, [94]K01610), malate dehydrogenase (maeB, [95]K00029); and
   downregulation of fructose-1,6-biphosphatase I (FBP, K03841), pyruvate
   phosphate dikinase (ppdK, [96]K01006), and ribulose-bisphosphate
   carboxylases (rubisco, [97]K01601–2) ([98]Figure 5 and [99]Table S4).
   Forty-three DEGs of starch and sucrose metabolism were upregulated
   including alpha-amylase (amy, [100]K01176), beta-amylase (bmy,
   [101]K01177), beta-glucosidase (bglX, K05349), invertase (sacA,
   [102]K01193), starch synthase (glgA, [103]K00703), sucrose phosphate
   synthase (SPS, [104]K00696), trehalose 6-phosphate phosphatase (otsB,
   [105]K01087), and trehalose 6-phosphate synthase (TPS, K16055).
   Additionally, SWEET transporters (SWEET, SWEET17; K15382) were highly
   upregulated but the H^+/sugar cotransporter (SUT) (SLC, K15378) was
   downregulated ([106]Figure 5 and [107]Table S4).

Figure 5.

   [108]Figure 5
   [109]Open in a new tab

   Effect of phytoplasma on sucrose accumulation in SCWL leaves and
   stalks. Heatmaps of upregulated and downregulated DEGs of sugar
   transporters, carbon fixation in photosynthetic organisms, starch and
   sucrose metabolism are shown. Blue arrow, upregulation; red arrow,
   downregulation.

   In stalks, carbon fixation in photosynthetic organisms was
   downregulated including GOT1, pecK, and ppdK. Seventeen DEGs of starch
   and sucrose metabolism were also suppressed including bmy, glgA, glgC,
   sucrose synthase (SUS, [110]K00695), and TPS, whereas beta-glucosidase
   (K05350), glucose-6-phosphate isomerase ([111]K01810), and otsB were
   upregulated ([112]Figure 5 and [113]Table S5). On the contrary to
   leaves, SWEET transporter, SWEET15 (K15382), was downregulated but SLC
   was upregulated ([114]Figure 5 and [115]Table S5).

2.4.3. Regulation of Genes Related to Plant–Pathogen Interactions

   Most DEGs in plant–pathogen interaction pathways were upregulated
   ([116]Figure 6 and [117]Table S4) including calmodulin (CALM,
   [118]K02183), cathepsin F (CTSF, [119]K01373), cyclic nucleotide-gated
   channel (CNGC, K05391), disease resistance proteins [RAR1 (K13458),
   RPM1 (K13457), and RPS2 (K13459)], glycerol kinase (GK, [120]K00864),
   mitogen-activated protein kinase kinase (MAP2K1, K04368; MKK4/5,
   K13413), MAP kinase substrate 1 (MKS1, K20725), and
   pathogenesis-related genes including transcriptional activator
   pattern-triggered immunity (PTI5, K13433), pathogenesis-related protein
   (PR1, K13449), and WRKY transcription factors (WRKY2, K18835; WRKY22,
   K13425; WRKY33, K13424). Downregulated DEGs included calcium-dependent
   protein kinase (CPK, K13412), elongation factor Tu (tuf, [121]K02358),
   and 3-ketoacyl-CoA synthase 11 (KCS, K15397). In addition, one of the
   plant defense-related genes, invertase ([122]K01193), was accelerated
   during sugarcane–phytoplasma interactions along with callose synthases
   (K11000) ([123]Table S4). In stalks, upregulated DEGs included RPM1,
   KCS, WRKY22, WRKY24, and WRKY33, while CALM, CNGC, CPK10, and PR1 were
   downregulated ([124]Table S5).

Figure 6.

   [125]Figure 6
   [126]Open in a new tab

   Effect of phytoplasma on plant–pathogen interaction in SCWL sugarcane
   leaves and stalks. Heatmaps of upregulated and downregulated DEGs of
   the plant–pathogen interaction are shown. Blue arrow, upregulation; red
   arrow, downregulation.

   KEGG pathway analysis of SCWL leaves revealed 113 DEGs encoding
   transcription factors (TFs) which can be categorized into six types
   ([127]Table S6). The largest category was helix-turn-helix proteins
   including MYB (MYBP, K09422), and MYB-related TF LHY (LHY, K12133). MYB
   TFs were upregulated along with other TFs including TGA (TGA, K14431),
   phytochrome-interacting factor 4 (PIF4, K16189), teosinte
   branched1/cycloidea/proliferating cell factors (TCP, K16221), and WRKYs
   ([128]Table S6). In stalks, TFs such as MYB, TGA, WRKY22, and WRKY33
   were upregulated, while PIF4 was downregulated ([129]Table S7).

2.4.4. Regulation of Genes Related to Signal Transduction by Plant Hormones

   In SCWL leaves, most DEGs of the plant hormone signal transduction
   pathway were altered ([130]Table S4), including upregulated DEGs of
   abscisic acid (ABA) [e.g., ABA receptor PYR/PYL family (PYL, K14496),
   ABA responsive element binding factor (ABF, K14432), catalase (CAT,
   K03781), and MAPK kinase kinase (MAPKKK17/18, K20716)], ethylene (ETH)
   signaling pathway [e.g., ethylene receptor (ETR, K14509),
   ETH-responsive TF 1 (ERF1, K14516), endochitinase B (ChiB, K20547), and
   MAPK kinase (MKK4/5, K13413; MKK9, K20604)], auxin (AUX) (AUX1, K13946;
   AUX/IAA, K14484; GH3, K14487; SAUR, K14488), cytokinin (CK) (A-ARR,
   K14492; AHP, K14490; HK, K14489), gibberellin (GA) (RGA, K14494 and
   PIF4), jasmonic acid (JA) (COI1, K13463; JAZ, K13464), salicylic acid
   (SA) [PR1, K13449 and TGA TF (K14431)], and brassinosteroid (BR) (TCH4,
   K14504). On the contrary, A-ARR of CK, PIF4 of GA, and PR1 of SA in
   stalks were suppressed, whereas JAR1 and JAZ of JA and NPR1 of SA were
   upregulated ([131]Table S5).

2.4.5. Regulation of Genes Related to Secondary Metabolites

   In leaves, key genes in flavonoid biosynthesis were downregulated
   ([132]Table S4), including chalcone synthase (CHS, [133]K00660),
   caffeoyl-CoA O-methyltransferase (CCoAOMT, [134]K00588), chalcone
   isomerases (CHIL, [135]K01859), flavonoid 3,5-hydroxylase (CYP75A,
   K13083), and flavonoid 3-monooxygenase (CYP75B1, K05280), while some
   were also downregulated in stalks ([136]Table S5). Genes in
   phenylpropanoid biosynthesis were upregulated in SCWL leaves
   ([137]Table S4), including cinnamyl-alcohol dehydrogenase (CAD,
   [138]K00083), cinnamoyl-CoA reductase (CCR, K09753), coniferyl-aldehyde
   dehydrogenase (REF1, K12355), peroxidase (POD, [139]K00430),
   phenylalanine ammonia-lyase (PAL, K10755), and shikimate
   O-hydroxycinnamoyltransferase (HCT, K13065), while some were found to
   be upregulated in stalks ([140]Table S5).

   In leaves, the DEGs of BR biosynthesis were upregulated ([141]Table
   S4), including typhasterol/6-deoxotyphasterol 2-alpha-hydroxylase
   (CYP92A6, K20623), PHYB activation-tagged suppressor 1 (CYP734A1,
   K15639), steroid 22-alpha-hydroxylase (DWF4, K09587), and steroid
   5-alpha-reductase (DET2, K09591), while some were also upregulated in
   stalks ([142]Table S5). In SCWL leaves, the DEGs of diterpenoid
   biosynthesis were upregulated ([143]Table S4), including ent-kaurene
   synthase (GA2, K04121), ent-kaurenoic acid monooxygenase (KAO, K04123),
   GA 2-beta-dioxygenase (GA2ox, K04125), and GA-44 dioxygenase (GA20ox,
   K05282), whereas some from stalks were downregulated including
   ent-kaurene oxidase (GA3, K04122), GA2ox and GA20ox ([144]Table S5).
   Monoterpenoid, sesquiterpenoid, and triterpenoid biosynthetic genes
   including germacrene D synthase (K15803) and NAD+-dependent farnesol
   dehydrogenase (FLDH, K15891) were upregulated in both leaves and stalks
   ([145]Tables S4 and S5).

   In leaves, the DEGs of carotenoid biosynthesis including
   abscisic-aldehyde oxidase (AAO, K09842), abscisic acid 8′-hydroxylase 2
   (CYP707A, K09843), β-carotene 3-hydroxylase (crtZ, K15746), carotenoid
   epsilon hydroxylase (LUT1, K09837), 9-cis-epoxycarotenoid dioxygenase
   (NCED, K09840), and xanthoxin dehydrogenase (K09841) were downregulated
   but abscisic-aldehyde oxidase was upregulated ([146]Table S4). In
   stalks, AAO was upregulated, while β-carotene isomerase and NCED were
   downregulated ([147]Table S5).

2.5. Verification of DEGs by Real-Time PCR

   To validate RNA-seq data, a total of twenty randomly selected candidate
   genes from leaves and stalks with upregulation and downregulation
   ([148]Table S8) were verified by real-time PCR. A comparison of RNA-seq
   with real-time PCR showed a correlation coefficient (R^2) of 0.795925.
   The results confirmed the expression trends of all candidate genes in
   accordance with the analysis of the transcriptome data ([149]Figure 7).

Figure 7.

   [150]Figure 7

   [151]Figure 7
   [152]Open in a new tab

   Validation and comparison of randomly selected DEGs of RNA-seq data
   (white bar) and real-time PCR quantification (black bar) of leaves (A),
   stalks (B) and correlation coefficients (R^2) between RNA-seq and
   real-time PCR results for differential gene expression in leaves and
   stalks of asymptomatic and symptomatic sugarcane (C).

3. Discussion

   The pathogenicity of SCWL phytoplasma for sugarcane is still unclear.
   Since phytoplasmas are difficult to culture in laboratory conditions
   and in vitro infection is exceptionally impracticable [[153]2],
   12-month-old SCWL and asymptomatic Saccharum hybrid cv. KK3 were
   collected from the same field and used for investigation to simulate
   the natural condition. The maturation phase is an important stage for
   harvesting since it gives the highest sugar yield with an economically
   acceptable marginal return for the sugar industry [[154]9]. The
   phenotypic characteristics of SCWL sugarcanes including height, stalk
   diameter, and sugar content were significantly lower, which
   corresponded to a 10^4 times higher phytoplasma expression ratio
   detected than those of asymptomatic canes. Furthermore, new buds
   emerging from such SCWL seed canes generated SCWL symptoms of a white
   grassy shoot phenotype with chlorosis leaves [[155]6].

3.1. SCWL Phytoplasma Affects Chlorophyll Metabolism and Photosynthesis

   In SCWL leaves, NYC1 and HCAR that, respectively, reduce chlorophyll b
   (Chl b) and chlorophyll a (Chl a) in chlorophyll metabolism
   [[156]12,[157]13] were repressed similarly to that reported in
   grapevine leaves infected with Flavescence dorée (FD) phytoplasma
   [[158]14]. Moreover, hemA, hemC, hemE, hemL, and hemY involved in the
   formation of protoporphyrin IX, a key precursor of chlorophyll
   biosynthesis, were downregulated along with chlH, chlE and por
   ([159]Figure 4) that modify Mg^2+ branching and form an isocyclic
   pentanone ring of chlorophyll [[160]13,[161]15]. High expression of
   chlorophyllase additionally degrades chlorophyll by cleaving the phytol
   tail and removing magnesium from the porphyrin ring [[162]16,[163]17].
   Such downregulated mechanisms of porphyrin and chlorophyll gene
   expression together with the enhancement of chlorophyllase activity in
   leaves of SCWL sugarcane evidently explain the symptoms of leaf
   chlorosis and low chlorophyll content caused by SCWL phytoplasma. The
   findings agree with the growth limitation of phytoplasma-infected
   sesame plants where photosynthetic rates were reduced due to the
   depletion of chlorophyll content, photosystem II photoprotection, and
   photosynthetic capacity [[164]18,[165]19].

   In this work, photosynthesis-related genes were mostly downregulated in
   SCWL leaves, which agrees with the observation for phytoplasma-infected
   Chinese jujube leaves in which the Lhcb gene family and photosystem II
   were downregulated [[166]20,[167]21]. Such suppression was also found
   in genes associated with photosystem I and II, including the cytochrome
   b6/f complex and ATP synthase in phytoplasma-infected grapevine
   [[168]22,[169]23], periwinkle plant [[170]24], and coconut palm
   [[171]25]. As part of defense mechanisms in response to biotic stress,
   photosynthesis genes are globally downregulated [[172]26], which
   decrease phloem loading and carbohydrate accumulation of the infected
   leaves as a consequence [[173]23] ([174]Figure 4 and [175]Figure 5).

   The other downregulated pathway affecting photosynthesis,
   photoprotection, and signaling in SCWL sugarcanes was the carotenoid
   pathway. A reduction in carotenoid content was found in
   phytoplasma-infected apple, Chinese jujube, cranberry, grapevine, and
   Napier grass [[176]14,[177]20,[178]27,[179]28,[180]29]. Low levels of
   the antioxidant carotenoids in phytoplasma-infected plants have a
   profound effect on plant physiology and response to stress since
   carotenoids play important roles in protecting cell damage caused by
   ROS [[181]30], and protect chlorophyll and the photosynthetic apparatus
   from photodamage [[182]31]. Moreover, carotenoids are precursors for
   plant hormone ABA; they are involved in the stress response [[183]32]
   and work together with chlorophyll in the process of light harvesting
   during photosynthesis [[184]33]. Therefore, downregulation of
   carotenoid-related genes in SCWL sugarcanes must have a downstream
   effect on ABA levels, which impacts the ability of the plant to respond
   to the phytoplasma and reduces the efficiency of photosynthesis.

3.2. SCWL Phytoplasma Alters the Gene Expression of Sucrose Accumulation

   In SCWL leaves, several genes related to carbon fixation in
   photosynthetic organisms, such as GOT1, maeB, and pecK, were
   upregulated ([185]Figure 5) similarly to those in leaves of cranberry
   infected with phytoplasmas [[186]29], Paulownia fortunei [[187]34],
   pepper, and Ziziphus jujuba Mill [[188]35]. Moreover, ppdK that
   converts pyruvate to phosphoenolpyruvate (PEP) in the C4 photosynthesis
   pathway [[189]36] was negatively regulated in SCWL leaves similar to
   that of phytoplasma-infected Paulownia [[190]37]. In addition, rubisco
   was also downregulated like that of phytoplasma-infected Chinese
   jujube, indicating the limitation of photosynthesis imposed by the
   carboxylation [[191]20].

   In this study, the accumulation of sugar was negatively affected by
   SCWL phytoplasma by the upregulation of genes involved in starch and
   sucrose metabolism of leaves (source organ) such as alpha-amylase,
   beta-amylase, invertase, starch synthase, SPS and TPS ([192]Figure 5),
   similarly to those of phytoplasma-infected Chinese jujube [[193]21],
   grapevine [[194]22], and tomato [[195]38]. Such high expression of
   invertase, SPS, and TPS in SCWL leaves, but downregulation in stalks,
   suggests that sucrose transport is blocked and leads to a high sucrose
   content in leaves but low in stalks (the sink organ) [[196]38].
   Furthermore, such a block would affect sugar signaling and auxin
   crosstalk that play important roles in plant growth and development
   [[197]39] and result in thinner stalks compared to the asymptomatic
   sugarcanes. Likewise, a sugar transporter, of the SWEET family, that
   maintains the equilibrium between membranes [[198]40] was upregulated
   in SCWL leaves but downregulated in stalks ([199]Figure 5), which would
   lead to unbalanced carbohydrate content between source and sink organs,
   similarly to those reported in coconut palm [[200]41], periwinkle,
   tobacco [[201]42], and tomato infected by phytoplasmas [[202]43]. On
   the contrary, SUT that transports sucrose from the photosynthetic organ
   to the sieve element–companion cell complex was downregulated in SCWL
   leaves ([203]Figure 5), which is similar to phytoplasma-infected tomato
   and plants expressing SUT antisense RNA, in which the rate of sucrose
   exudation was lower in the infected plants [[204]43]. Moreover,
   phytoplasma possibly acts as an additional ‘sink’ to accumulate
   carbohydrates from photosynthesis and then blocks sugar transport from
   the leaf source to stalk sink [[205]41]. Altogether, the loading of
   sugar from leaves to stalks was disrupted, which resulted in lower
   sugar content in SCWL stalks than that of asymptomatic sugarcanes.

3.3. SCWL Phytoplasma Alters the Expression of Genes Related to
Plant–Pathogen Interactions

   Sugars are not only the primary component furnishing energy but also a
   structural material for defense responses in plants [[206]44].
   Pathogens utilize sugar for their survival, while plants adapt their
   sugar production to stimulate plant defense responses. Invertases are
   one of the sucrose hydrolytic enzymes for carbohydrate degradation
   [[207]45], and a key regulator for sucrose accumulation in sugarcane
   stalks [[208]46]. SUS that was upregulated in SCWL stalks is localized
   in both companion cells and sieve elements of phloem and supplies UDP
   glucose to induce callose plugs in the sieve pores [[209]47]. Such
   obstruction of the sieve tubes by callose deposition was previously
   reported in phytoplasma-infected C. roseus, Euphorbia pulcherrina
   [[210]48], and Vitis vinifera L. cv. Chardonnay [[211]23]. Moreover,
   invertase and callose synthases of SCWL sugarcanes were elevated in
   leaves, which may lead to sieve-tube occlusion, which is required to
   prevent the colonization of SCWL phytoplasmas. The results of this work
   are in accordance with those of phytoplasma-infected apple [[212]49]
   and grapevine [[213]23].

   Calcium-binding proteins such as calmodulin (CAM) and calcium-dependent
   protein kinase (CPK) are involved in the signaling network in plant
   innate immunity [[214]50]. CPK-CNGCs are cation transport channels
   regulated by CAM [[215]51]. Those genes were upregulated in
   phytoplasma-infected American cranberry [[216]29], P. fortunei
   [[217]52], and Mexican lime [[218]53], similarly to the results for
   SCWL sugarcanes. Upregulation of calcium signaling cascades sensed by a
   high expression of CALM in SCWL sugarcanes leads to Ca^2+ accumulation.
   This accumulation should induce the expression of CPK, followed by
   rboh, which is a main source for ROS and plays an important role in the
   plant disease response [[219]54]. Upregulation of rboh in SCWL
   sugarcanes implies that the pathogenic development of SCWL phytoplasma
   possibly required host Rboh to induce ROS and cell death, which
   resulted in the anchoring of SCWL disease. However, CPK expression in
   SCWL canes at the maturation phase was unexpectedly downregulated. The
   result would lead to a decrease in the activity of SOD, POD, and CAT,
   correlating with the silencing CPK in powdery mildew-infected wheat
   [[220]55] and CPK mutants of Arabidopsis that exhibited vigorous
   pathogen growth and development of disease symptoms [[221]56].
   Therefore, downregulation of CPK may be a mechanism whereby SCWL
   phytoplasma triggers sugarcane to allow for more invasions of the
   pathogen.

   MAPK cascades play important roles in the regulation of innate immune
   responses in plants [[222]57] and directly control gene expression by
   phosphorylating TFs [[223]58]. These genes were highly expressed in
   SCWL sugarcanes, indicating that infection by phytoplasma accelerated
   the expression of the MAPK signaling pathway and boosted the stress
   response in sugarcane. Furthermore, plant disease resistance genes
   involved in the innate immune response against pathogen invasion such
   as RAR1, RPS2, and PTI5 [[224]59] were upregulated in SCWL leaves,
   similarly to those found in witches’ broom disease (WBD) in Nerium
   indicum Mill [[225]60]. PTI5, a transcriptional activator of
   ETH-responsive element binding protein, was enriched in SCWL
   sugarcanes, which potentially induces the expression of CAT [[226]61].
   In concordance with our results, PR1 encoding a toxic protein against
   pathogen invasion [[227]62] was upregulated in periwinkle leaf
   yellowing (PLY) phytoplasma-infected periwinkle [[228]63], Ca. P.
   solani-infected tomato [[229]30], Ca. P. asteris-infected Arabidopsis
   [[230]64], Ca. P. mali-infected apple [[231]65], and Ca. P.
   solani-infected grapevine [[232]66]. Acceleration of GK in SCWL
   sugarcanes agrees with that of WBD in Nerium indicum L., which is
   involved in phytoplasma resistance [[233]67]. In this work, WRKY22 that
   modulates ETH, JA, and SA signaling [[234]68] was upregulated in SCWL
   sugarcanes. Upregulation of WRKY33 in SCWL sugarcanes is similar to
   that of Xanthomonas albilineans-infected sugarcanes [[235]69].
   Moreover, upregulation of PTI5 together with WRKYs in SCWL sugarcanes
   may activate the defense-related genes involved in phytoalexin
   production, which plays a crucial role in plant defense against
   pathogens and contributes to the overall resistance of the plant
   [[236]70]. Notably, some DEGs were upregulated in leaves but
   downregulated in stalks due to the fact that leaves are frequently
   exposed to pathogens and promptly activate defense mechanisms to
   prevent the invasion [[237]71], whereas stalks primarily serve as
   structural and nutrient transport rather than having a defense function
   [[238]72].

3.4. SCWL Phytoplasma Accelerates Plant Hormone Signaling Transduction

   Genes responsible for plant hormone signaling pathways were mostly
   upregulated in SCWL sugarcanes including ABA, AUX, BR, CK, ETH, GA, JA,
   and SA, which are involved in plant defense responses, particularly
   host–pathogen interactions [[239]73]. Similar to these results, ABA
   signaling pathway-related genes such as PYL, ABF, and NCED of
   phytoplasma-infected apple, Mexican lime, mulberry, and Paulownia
   fortunei were upregulated [[240]53]. Genes involved in the AUX
   biosynthesis of phytoplasma-infected Mexican lime [[241]53] and sesame
   [[242]32] were upregulated along with CK and GA-related genes, which
   are similar to those of SCWL sugarcanes in this work. Furthermore, SCWL
   phytoplasma caused the upregulation of genes involved in the ETH
   signaling pathway such as ERF and ETR, similar to those of Ca. P.
   solani-infected grapevine [[243]74], Ca. P. aurantifolia-infected
   Mexican lime [[244]53], and Paulownia witches’ broom
   phytoplasma-infected P. fortunei [[245]75]. Upregulation of ETH genes
   is associated with the activation of genes involved in the production
   of ROS, which are important components of the plant defense response
   [[246]22].

   Phytoplasma infection controls cell balance and plant development by
   secretion of the SAP11 effector that targets TCP TF, which, in turn,
   controls cell proliferation, cell maturation, plant development, and
   senescence by binding to the lipoxygenase (LOX) promoter in Ca. P.
   asteris AY-WB-infected Arabidopsis [[247]76] and mediates the
   production of oxylipin, a precursor of JA biosynthesis [[248]77]. TCP5
   binds directly to the promoter of the PIF4 gene to increase its
   expression level and interacts with the PIF4 protein [[249]78]. In
   tomato and grapevine infected with Ca. P. solani, LOX was upregulated
   [[250]30,[251]79]. In this work, TCPs were upregulated in SCWL leaves
   and resulted in an enhancement of the expression of LOX, which may lead
   to increased levels of JA in sugarcanes. Accumulation of JA can in turn
   trigger the expression of the downstream JA-responsive genes involved
   in the plant defense against SCWL phytoplasma. Upregulation of such
   genes in ‘Ca. P. asteris’ strain AY-WB-infected Arabidopsis was
   similarly observed [[252]76].

   The expression of several SA-related genes such as PR1 was upregulated
   in SCWL sugarcane, similarly to those of phytoplasma-infected apple,
   Arabidopsis, grapevine, Madagascar periwinkle, and tomato
   [[253]30,[254]64,[255]65,[256]80,[257]81]. The PR1 regulates gene
   expression by interacting with TGA TF that interacts with the promoter
   of PR itself in the presence of SA [[258]82]. Thus, the induction of
   PR1 expression would promote the biosynthesis of the plant SA
   [[259]83]. Upregulation of TCH4 in SCWL sugarcane is similar to that of
   phytoplasma-infected paulownia [[260]84], which is likely to be
   involved in the biosynthesis of hemicellulose, a complex carbohydrate
   component of plant cell walls [[261]85]. Together with the
   downregulation of BRI1, these results suggest a potential plant defense
   mechanism by sugarcanes against SCWL phytoplasma.

3.5. SCWL Phytoplasma Alter the Expression of Genes Involved in Secondary
Metabolites

   Phenylpropanoid precursors including benzenoids, coumarins, flavonoids,
   hydroxycinnamates, and lignin are involved in plant development and
   plant–pathogen interactions relating to environmental stress and
   disease tolerance [[262]86]. In this study, CAD, CCR, 4CL, PAL, and POD
   (which play roles in the phenylpropanoid pathway) were upregulated in
   SCWL sugarcanes, leading to an increase in lignin content [[263]87]
   that would be related to strengthening the cell walls of sugarcane in
   response to an attack by phytoplasma. Furthermore, several upregulated
   MYB TFs that are responsible for the regulation of biosynthetic genes
   for phenylpropanoid and lignin were detected in SCWL sugarcanes like
   those reported in Arabidopsis [[264]88]. Therefore, the contents of
   various phenylpropanoid compounds including lignin, flavonoids, and
   coumarins, which play crucial roles in the resistance against
   phytoplasma infection [[265]89], were potentially increased in SCWL
   sugarcane, acting as antioxidants to protect sugarcane from phytoplasma
   infection.

   Flavonoids and isoflavonoids act as antioxidants to protect plants from
   pathogens by boosting growth and development [[266]90]. The
   downregulation of CHS and CYP75B1, which are key genes of flavonoid
   biosynthesis, in SCWL sugarcane suggests a reduction in flavonoid
   accumulation, which, in turn, could contribute to an escalation in
   disease symptoms when the plant is exposed to a pathogenic challenge
   [[267]91]. Genes related to diterpenoid biosynthesis in
   phytoplasma-infected lime were upregulated including GA2, GA3, KAO,
   GA2ox, and GA20ox [[268]53] like those found in SCWL sugarcanes in this
   study. Diterpenoids exhibit a species-specific chemical defense against
   pathogens [[269]92]. A common precursor of diterpenoids, geranylgeranyl
   diphosphate, also contributes to GA biosynthesis [[270]93]. Moreover,
   upregulation of sesquiterpenoid and triterpenoid biosynthesis in SCWL
   sugarcane may increase β-caryophyllene, δ-elemene, and germacrene D,
   similarly to those phytochemicals found in phytoplasma-infected
   Hypericum perforatum L. [[271]94]. The increase in sesquiterpene
   biosynthesis in SCWL sugarcanes may attract the insect vector to
   disperse SCWL phytoplasma as suggested for AP phytoplasma-infected
   apple and tobacco [[272]95]. Hence, changes in the biosynthesis of
   flavonoids, terpenoids, and sesquiterpenes in SCWL sugarcane may
   constitute a component of the pathogen’s strategy for disease
   dissemination.

4. Conclusions

   SCWL sugarcanes at the maturation phase harbor 10^4 times more
   phytoplasma than asymptomatic plants growing in the same field and have
   smaller stalk diameters, shorter height, and lower sugar content.
   Transcriptomic analysis revealed alterations in gene expression for
   several pathways in response to an infection by phytoplasma including
   photosynthesis, porphyrin and chlorophyll metabolism, starch and
   sucrose metabolism, plant hormone signaling transduction, flavonoid
   biosynthesis, and plant–pathogen interactions. Suppression of porphyrin
   and chlorophyll metabolism coupled with an increase in chlorophyllase
   expression led to a reduction in chlorophyll levels and in
   photosynthesis that corresponded to the white leaf phenotype.
   Enhancement of sugar transporters in leaves, but suppression in stalks,
   suggests that sucrose transport is blocked and results in lower sucrose
   levels in stalks. Upregulation of SUS leads to a UDP-glucose-induced
   callose plug formation in sieve pores that serves as a defense
   mechanism against colonization by phytoplasma. The expression of genes
   associated with calcium cascades led to Ca^2+ accumulation, which
   contributed to plant immunity. However, downregulation of CPK would
   result in a reduction in SOD, POD, and CAT activities, consequently
   facilitating an invasion by the pathogen. Furthermore, enrichment of
   signaling transduction pathways (e.g., MAPK, CAT, PR1, GK, and
   transcription factors) and plant hormone signaling transduction (e.g.,
   ABA, AUX, CK, GA, JA, SA, and ETH-responsive genes) positively promotes
   the defense response of sugarcane against phytoplasma. It is speculated
   that phytoplasma secretion effectors might target TCPs and cause an
   expression of LOX and subsequently increase the JA level. Upregulation
   of the phenylpropanoid pathway led to lignin accumulation in response
   to an attack by phytoplasma, while the expression of sesquiterpene
   biosynthesis possibly attracts the insect vectors for transmission,
   thereby facilitating the spread of phytoplasma to other sugarcanes.
   However, downregulation of flavonoid biosynthesis would potentially
   intensify the SCWL symptoms upon challenge by phytoplasma. These SCWL
   sugarcane transcriptomic profiles represent the first comprehensive
   view of genes and pathways involved in the response of sugarcane
   towards infection by phytoplasma, which will facilitate in finding
   specific corresponding marker genes for further sustainable development
   of disease prevention, protection, and management.

5. Materials and Methods

5.1. Sugarcane Samples and Plant Growth Parameters

   Saccharum hybrid cv. KK3 was grown in a field of the MitrPhol sugarcane
   plantation, Nong Kung Si district, Kalasin province, Thailand
   (16°42′56.0″ N 103°22′34.2″ E). Treatments of organic fertilizers in
   January and chemical fertilizers in February [N-P-K = 21-7-18, 312.5
   kg/ha April (N-P-K = 46-0-0, 156.25 kg/ha and 21-7-18, 156.25 kg/ha),
   and July (N-P-K = 21-7-18, 312.5 kg/ha)] were applied. Five of the
   12-month-old symptomatic and asymptomatic SCWL sugarcanes were randomly
   harvested from the same location. Phenotypic characteristics of SCWL
   sugarcanes were determined visually based on the basic paler leaves
   compared to the asymptomatic ones. Sugarcane stalks of the 14th–16th
   internode from the root and 1st–3rd leaves from shoots were immediately
   sampled between 11 a.m. and 4 p.m. and kept in an RNA storage reagent
   (Tiangen, Beijing, China) at −20 °C until use. Plant growth parameters
   including height and stalk diameter were measured. Sugar content was
   analyzed using a master refractometer (ATAGO, Tokyo, Japan) and
   recorded as a Brix percentage.

5.2. RNA Extraction

   Both stalk and leaf samples were cut into small pieces and immediately
   frozen with liquid nitrogen and ground to a fine powder. Total RNA was
   extracted using a GF-1 total RNA extraction kit (Vivantis, Shah Alam,
   Malaysia), as described by the manufacturer. RNA quality and quantity
   were analyzed using a Nanodrop ND-100 (Thermo Scientific, Waltham, MA,
   USA).

5.3. Detection of Phytoplasma in Sugarcane Using Real-Time PCR

   DNA samples of SCWL sugarcane were prepared using the CTAB method
   [[273]96]. The phytoplasma 16S rRNA gene was amplified by nested PCR
   using primers described previously [[274]48]. The PCR master mix
   contained 100 ng DNA template, 1× Phusion HF buffer, 200 µM dTNPs, 0.5
   µM each primer, and Phusion Hot Start II DNA polymerase (0.02 U/µL)
   (Thermo Scientific, Waltham, MA, USA). Sugarcane 18S rRNA gene was
   amplified using 18S rRNA-F and 18S rRNA-R primers [[275]97] following
   the PCR reaction above. The purified PCR products were sent for
   sequencing at Macrogen (Seoul, Republic of Korea). The numbers of
   phytoplasma 16S rRNA and sugarcane 18S rRNA genes were calculated using
   the formula of Staroscik [[276]98] to generate standard curves.

   To determine the number of active phytoplasma in sugarcanes, total RNA
   was converted to cDNA using random hexamers and RevertAid First strand
   cDNA synthesis (Thermo Scientific, Waltham, MA, USA). Real-time PCR was
   performed using a Master Cycler Realplex 4 (Eppendorf, Hauppauge, NY,
   USA) with a KAPA SYBR FAST qPCR master mix (Kapa Biosystems, Seoul,
   Republic of Korea) with reaction conditions: 95 °C for 3 min, 40 cycles
   at 95 °C for 10 s, and 62 °C for 1 min. Quantification of 16S rRNA
   genes of phytoplasma and 18S rRNA genes of sugarcane was calculated
   based on the standard curve using comparative cycle threshold (Ct)
   values [[277]99]. The content of phytoplasma in each sample was
   normalized using the corresponding sugarcane 18S rRNA gene.

5.4. RNA Sequencing and Transcriptome Analysis

   Total RNA samples of 3 biological replicates of symptomatic SCWL leaves
   (SL), asymptomatic leaves (AL), symptomatic SCWL stalks (SS), and
   asymptomatic stalks (AS) were sent for RNA sequencing at NovogeneAIT
   Genomics Singapore Pte (Singapore). Briefly, RNA samples with an RNA
   integrity number (RIN) of more than 6.5 were subjected to rRNA
   depletion, mRNA random fragmentation, cDNA construction, and terminal
   end ligation with poly-A and sequencing adaptors. Then, cDNA libraries
   were size selected and quantified before sequencing using an Illumina
   Hiseq X instrument (2 × 150 bp) (Illumina, San Diego, CA, USA).

   Raw reads were uploaded to Galaxy Project (usegalaxy.org) and initially
   were quality checked using FastQC [[278]100]. Low-quality sequences at
   Q20 and adapter contamination were filtered out using Trimmomatic
   [[279]101]. The clean reads were mapped to the monoploid sugarcane
   genome reference ([280]https://sugarcane-genome.cirad.fr; accessed on
   14 February 2020) using Bowtie2/TopHat [[281]102]. The mapped reads
   were assembled using Cufflink, merged using Cuffmerge, and performed
   DEGs using Cuffdiff [[282]103]. Principal component analysis (PCA) was
   performed using DESeq2 [[283]104].

   DEGs were cut off at log[2] FC ≥ 1 with a false discovery rate (FDR) of
   <0.05. Gene function was categorized using gene id and protein products
   from the gene ontology (GO) [[284]11] and general feature format (GFF)
   annotation file provided by the reference genome sequence [[285]11]. GO
   terms of all DEGs were subjected to singular enrichment analysis (SEA)
   in agriGO [[286]105]. The GO term enrichment was calculated by
   comparing the frequency of gene sets with different GO terms relative
   to reference gene sets of Plant GO slim using Fisher as statistical
   test method, Benjamini–Yekutieli as a multi-test adjustment method, and
   5 as a minimum number of mapping entries [[287]106]. Then, biological
   pathways and molecular interaction networks of DEGs were classified by
   mapping amino acid sequences using GhostKOALA and KEGG mapper
   [[288]107,[289]108]. Pathway enrichment analysis was performed using
   the piano in R package based on geneSetStat “reporter” [[290]109].
   Pathways with p values of <0.05 were considered as significantly
   enriched pathways.

5.5. Quantification of Candidate Genes by Real-Time PCR

   One microgram of total RNA was used for cDNA synthesis with oligodT
   primer using RevertAid First strand cDNA synthesis (Thermo Scientific,
   Waltham, MA, USA). The candidate genes were randomly selected from the
   significantly enriched pathways and primers were accordingly designed
   using Primer BLAST [[291]110] ([292]Table S8). Real-time PCR was
   performed in 3 biological and 3 technical replicates using a Master
   Cycler Realplex 4 (Eppendorf, Hauppauge, NY, USA) with KAPA SYBR FAST
   qPCR master mix (Kapa Biosystems, Seoul, Republic of Korea) using
   reaction conditions: 95 °C for 3 min, 40 cycles at 95 °C for 10 s, and
   59 °C for 1 min. Relative levels of the expression of DEGs of SCWL and
   asymptomatic sugarcanes were determined using the ΔΔC[T] method
   [[293]111] with the GAPDH gene used as an internal control for data
   normalization.

5.6. Statistical Analysis

   Plant growth parameters were analyzed by t-test to determine
   statistical significance between groups at p-values of <0.05 by
   Microsoft Excel data analysis.

Acknowledgments