First molecular insights into the infection process provoked by Neofusicoccum parvum in Liquidambar styraciflua and the identification of new cysteine rich proteins in both organisms CURRENT STATUS: UNDER

Background Neofusicoccum parvum belongs to Botryosphaeriaceae family that groups endophytic and latent pathogens of woody plants responsible of diseases such as cankers, dieback and blight. Is a widespread pathogen in a broad host range including agricultural, horticultural and forestry plants, therefore is relevant to characterize molecular mechanisms involved in the disease. This work, report for first time a N. parvum as a pathogen of Liquidambar styraciflua . We established an in vitro pathosystem using foliar tissue in order to characterize the infection process through scanning electron microscopy. Because cysteine rich proteins (CysRPs) are well described for their important functions under plant-pathogen interaction, new CysRPs were identified for these organisms, and mRNAs expression of these proteins was analyzed at early times during the interaction. CFEM:common in several fungal extracellular membrane; CysRPs:cysteine rich proteins; DAB:diaminobenzidine; dpi:days post inoculation; ET:ethylene; FCRTP:fungal cystine rich transmembrane protein; GST1:glutathione S-transferase-1; JA:jasmonic acid; LTP:lipid transfer protein; LsCysRPs: Liquidambar styraciflua cysteine rich proteins; mRNAs:messenger RNA; NpCysRPs: Neofusicoccum parvum cysteine rich proteins; PR-1:pathogenesis related protein 1; ROS:reactive oxygen species; SA:salicylic acid; SCR96:small cysteine rich 96; SEM:scanning electron microscopy; SBN:Santuario del Bosque de Niebla; SsSSVP1:small secreted virulence-related protein in Sclerotinia sclerotiorum .

Until now there is no effective control strategies against N. parvum and is inferred that its transmitted horizontally [23] leading to a higher risk in a larger number of hosts, therefore it is relevant to characterize molecular mechanisms involved in the disease establishment in other important hosts, that is the case of Liquidambar styraciflua, a deciduous timber tree often used for reforestation, agroforestry, and landscaping [24]. Liquidambar styraciflua L. (Atingiaceae) is native from Americas and currently is distributed in regions localized in North and Central America [25] also has been widely introduced in eastern and central China [26]. Is also called American sweetgum and is an attractive hardwood species for potential bioenergy production [27]. This work report for the first time that N. parvum is a pathogen of L. styraciflua and through molecular approaches present knowledge of this interaction. In this sense, the objectives of this work were 1) describe for first time that N.
parvum is a pathogen of L. stytracifula, 2) to stablish an in vitro and reproducible pathosystem, 3) characterize the infection process by scanning electron microscopy (SEM) and 4) identify cysteine rich proteins (CysRPs) of both organisms that may be important during the defense response and infection process. Recent evidence indicates that pathogens effector proteins are small secrete cysteine rich proteins with a N-terminal signal peptide sequences, contain ≤ 300 amino acids and ≥ 2% of cysteine content [28,29]. Some currents examples are SCR96 of Phtytophtora cactorum [30] and SsSSVP1 in Sclerotinia sclerotiorum [31] where their silencing reduce the pathogen virulence respectively.
However, the evidence also indicates that effector proteins are highly specific and inherent of each pathogen, and no information has been generated about the CysRPs in any specie of the Botriosphariaceae family. Besides, CysRPs in plants are important components of the plant innate immune system, some examples include the α-defensins, lipid transfer proteins (LTPs), thionins, hevein-and knottintype peptides and cyclopeptide alkaloids [32].
In this work, a novel pathosystem is achieved and a first approach is made in the knowledge of CysRP in both actors of the interaction.

Results
Identification of N. parvum as a pathogen of L. styraciflua Eleven fungi were isolated from L. styraciflua leaves with disease visible symptoms such as necrosis and discoloration. Its potential pathogenicity was tested in L. styraciflua leaves and in seedlings of the plant model Arabidopsis thaliana (Figs. 1 and S1). The pathogenicity screening identified the fungal Liqui 1-3 strain as the most pathogenic, since in A. thaliana seedlings the fungus covers all the plant tissues provoking a notorious leaves discoloration at 7 dpi, L. styraciflua leaves developed clear necrosis and discoloration including the principal veins and the petiole at 8 dpi. Liqui 1-02, Liqui 2-2 and Liqui 2-3 triggered clear disease symptoms in L. styraciflua, but in contrast with the effect produced by Liqui 1-3, no noticeable change was observed in petiole coloration. In A. thaliana seedlings, these three isolates did not have a pathogenic critical effect, for example, while Liqui 2-2 covered more than 50% of the seedlings, the foliar tissue showed a greater vigor compared to the control. Liqui 1-2-01, Liqui 1-2-03 and Liqui 3 − 2 at 7 dpi showed a discrete pathogenic effect. A.
thaliana during the interaction with these isolates, developed a primary root with shorter length but increased number of secondary roots. The isolates Liqui 1-04, Liqui 1-01, Liqui 3-3 and Liqui 3 − 1 neither in L. styraciflua leaves nor in A. thaliana seedlings caused an effect.
To identified at molecular level the Liqui 1-3 strain, universal and specific primers were used (Table   S1, Fig. S2). The analysis revealed that Liqui 1-3 belongs to the Botryosphaeriaceae family as a Neofusicoccum parvum species.
Establishment of the L. styraciflua -N. parvum pathosystem Because we were interested in study the interaction between L. styraciflua and N. parvum we established the pathosystem using L. styraciflua leaves. We noticed that the infection progressed rapidly since at 1 and 3 dpi the inoculated leaves displayed brownish discoloration that was accentuated through time (Fig. 2), was notorious the petiole necrosis at 8 and 16 dpi, and the presence of whitish mycelium in the necrosis leaf area also was evident. To characterize the infection process in more detail, we analyzed the infected an uninfected tissue using SEM (Fig. 3). SEM images revealed that the fungus growth deeply in the leaf (adaxial) surface forming hyphae mass and leading to tissue degradation, the cuticle and the wax integrity was compromised ( Fig. 3a-d). Besides, the infection provoked petiole degradation. The transverse cut of the leaf base showed that the fungus was able to develop pycnidia, an asexual reproductive structure. The pycnidia were appear individually or as aggregates immersed in the plant tissue with thick walls that are composed of numerous cells (Fig. 3i-l). The longitudinal section of pycnidium show mature conidia. The conidiogenic cells without septa and with oval shape are localized perpendicular to the walls of the pycnidium. Because the Botryosphaeriaceae family members are characterized to be woody pathogens we tested the pathogenicity of Liqui 1-3 strain in fresh stems of L. styraciflua. As is shown in Fig S3, N. parvum at 7 dpi triggered notorious symptoms of disease such as discoloration and necrosis that covered a zone beyond the site of inoculation.
Detection of hydrogen peroxide in L. styraciflua leaves at early times of the infection process Its already known that reactive oxygen species (ROS) accumulate in plants cells during pathogen infection and may cause oxidative damage to proteins, DNA, and lipids or also act as signaling molecules to regulate defense response [33,34]. One of this species is the hydrogen peroxide and by DAB staining we detected clearly the presence of a dark brown precipitate in the infected leaves at early times (1 and 3 dpi) thus showing the detection of H 2 O 2 ( Fig S4).
Identification of CysRPs in L. styraciflua and N. parvum and their general features Cysteine rich proteins (CysRPs) are well described for their important functions under plant-pathogen interaction, and with the purpose of identify CysRPs in L. styraciflua and N. parvum, two databases with transcriptomic and genomic information respectively were analyzed (see Materials and Methods).
For each organism, five sequences encoding CysRPs were identified (LsCysRP1-5 and NpCysRP1-5) in all of them were recognized a putative start and stop codon with the exception of LsCysRP3 sequence where a stop codon was not localized (Table 1 and Table S2). The amino acid sequence length varies between 95 and 204, the molecular weight range among 7.7 and 17.5 KDa, also the cysteine content was determined. Curiously, with the exception of LsCysRP1 that had a calculated isoelectric point (pI) of 6.04, all the calculated LsCysRPs pIs were upper than 8.67, meanwhile to the exception of NpCysRP1 that had a pI of 7.57, all the calculated NpCysRPs pIs were lower to 5.48 (Table 1). To predict SS-bonds in L. styraciflua and N. parvum CysRPs, the servers Cyscon, Disulfind, DiANNA, CYS_REC and SCRATCH were used. The results revealed that LsCysRP1, LsCysRP2, LsCysRP3, LsCysRP4, LsCysRP5 have the potential to form 3, 4, 4, 0, and 6 disulfide bonds respectively. NpCysRP3, NpCysRP4 NpCysRP5 respectively. A notorious discrepancy about the selection of which cysteines form the pairs is detected between the methods (Table S3).
In order to know if the CysRPs have the potential to be secreted, an analysis with the TargerP-2.0 server was conducted. All sequences with the exception of LysCysRP2 have a peptide signal between 17 to 27 amino acids length. For corroborate these results, additionally analyses were performed using Protter and DeepLoc-1.0 servers, the results indicated that all CysRPs have a signal peptide and are extracellular proteins ( Table 2).
For identify possible functions and similarity regions in CysRPs, BLAST and MOTIF tools were used. No significant similarity was found for LsCysRP1, 2 and 4, neither for NpCysRP1, 2 and 3. In contrast, LsCysRP3 showed similarity with a lipid transfer protein and LsCysRP5 with gibberellin-regulated protein 1-like. Interestingly, for both NpCysRP4 and 5were identified a CFEM domain ( Table 1, Fig. 6S).

Cysrps Phylogenetic Analyses
To obtain more information about NpCysRPs, the corresponding phylogenetic analysis for each one however the cysteines remain at the same site into the sequences (Fig. S5). Other species were included in the alignment as Asperguillus leporis and Rhizoctonia solani, however, they have a smaller cysteines number and lower identities.
In addition with the Botryosphaeria lineage, NpCysRP2 phylogenetic tree contain orthologs of family Hypocreaceae, order Hypocreales including seven different species of Trichoderma genus with identity that ranged from 33.04 to 38.18%. Also, members of family Glomerellaceae were included such as species of the well-known Colletotrichum phytopathogenic genus such as the species asianum, nymphaeae and orchidophillum with 33.04, 36.28 and 37.19% amino acid identity respectively. Interesting, in the tree is also showed species of the order Sordariales as the human pathogen Madurella mycetomatis with 33.90% identity. After performed the alignment is revealed that NpCysRP2 introduces a new cysteine rich domain with the follow consensus motif C 1 [Y/F]xPx 9 − 10 C 2 x 6−8 C 3 C 4 x 4 C 5 x 2 Nx 2 C 6 x 10 − 23 C 7 Tx 8 C 9 x 3 C 10 at the N-terminal. Also, the multiple alignment of NpCysRP2 with the ortholog sequences of L. theobromae (67.52% identity) and D. corticola (62.07% identity) showed that NpCysRP2 is a larger protein since the L. theobromae and D. corticola sequences contain 310 and 296 amino acid respectively ( Fig S5X). Interesting, the proteoforms of L. phaseolina with a 78.07% identity while NpCysRP5 showed an 83.01% identity with L. theobromae.
Cenococcum geophilumn and Glonium stellatum belong to Gloniaceae family and present orthologs for both NpCysRP4 and NpCysRP5. Interesting, for NpCysRP4 were identified various orthologs inside Nectriaceae family, order Hyporcreales represented by the well-known phytopathogenic Fusarium species as verticillioides and oxysporum that were not found in NpCysRP5 phylogenetic tree using 33 sequences. In the case of NpCysRP5 a clear clade was identified represented by order Eurotiales where different Penicillium species were group with identities that ranged from 34.3 to 41.78%.
In the case of L. styraciflua CysRPs, no similar sequences were found for LsCysRP1, 2 and 4 therefore it was no possible to construct the corresponding phylogenetic trees. Expression of CysRPs mRNAs of L. styraciflua and N. parvum during early times of the infection process.
To explore if transcription of CysRPs mRNAs of L. styraciflua and N. parvum was modulated at early times of the interaction, quantitative PCR tests were performed. In Finally, our experimental design allowed us to compare the expression of NpCysRPs transcripts between 1 and 3 dpi. Figure 6B showed that there was an increment in the expression of NpCysRP1, 2 and 5 mRNAs being the last one which showed the more significantly increased. In contrast, NpCysRP4 showed a significant decrement.

Discussion
Liquidambar styraciflua L. (Atingiaceae) is used for reforestation and landscaping issues and has been proposed as an attractive hardwood species for potential bioenergy production [27]. Because of its importance, its relevant to attend the causes that limit integrity i.e. the phytosanitary problems. In general, is assumed that L. styraciflua has associated scarce pathogens [27,38] In this sense, information collected by Hepting in 1971 [39] enlisted a Cercospora liquidambaris, Septoria liquidambaris, Exosporium liquidambaris, Leptothyriella liquidambaris and Gloesporium nervisequm as common foliar pathogens of the sweetgum. In the other hand, L. theobromae and Botryosphaeriae dothidea have been identified responsible to provoke stem cankers and dieback in seedlings ubicated in nurseries and outplantings in the USA [38,39]. Currently, there are no new information about the nature of the pathogenic fungi infecting L. styraciflua, but prevalence the incidence of symptoms associated with diseases. Here, we reported that N. parvum is associated with L. styraciflua foliar damage and the pathosystem established in this work identified the Liqui 1-3 as a very aggressive strain since provoke several symptoms at early times post infection (Figs. 1 and 2). SEM analysis showed that the fungus was able to growth and develop pycnidium in order to produce a large amount of mature spores increasing its infective potential (Fig. 3). Our results are congruent with the reported for other Botryosphaeria species. Amponsah and colleagues [40] reported during the interaction of grapevine and N. luteum a faster rate (3 h after inoculation) of conidial germination on detached and wounded leaf and shoot surfaces than on attached and non-wounded leaf surfaces, indicating that conidium adhesion, germination and development were affected by the condition of the host plants. Also, in mamey sapote stem cuttings infected with L. theobromae at 30 dpi was possible to identified fruiting bodies immersed in the tissue [41]. It will be interesting in future works to characterize the Liqui 1-3 strain in more detail about its capacity to produce lytic enzymes and secondary metabolites with phytotoxic effect in comparison with other strains isolated from other ecological niches. Finally, it should not take aside the identification of other fungi isolated from L. styraciflua that also have pathogenic potential such as Liqui 1-02 and Liqui 2-2 ( Fig. 1 and Fig S1).
In addition to the phenotypic effects associated with pathogenicity caused by N. parvum in L.
styraciflua, also the identification of cysteine rich proteins was considered as a part of the molecular events triggered by both organisms during infection and defense response. Five CysRPs were identified for each organism (Table 1) and bioinformatic analyses revealed that LsCysRP1, 2 and 4 are proteins with unknown function suggesting that are species-specific proteins. LsCysRP3 was recognized as lipid transfer protein (LTP), this protein is consider an antifungal protein classified as PR-14 [42] with not yet known mode of action, but in vitro conditions has the ability to enhance the intermembranal exchange causing a posteriori the fungal cell death [32], in barley leaves inhibit the growth of F. solani [43], while transgenic Populus tomentosa over-expressing a LTP of Leonurus japonicus was resistant to A. alternate and C. gloesporioides [44]. LsCysRP5 was identified as gibberellin-regulated protein 1-like, named GASA family in A. thaliana and Snakin in Solanum tuberosum. This class of proteins form 5 or 6 disulfide bonds that are necessary for the structure, in consequence for the protein function [45], moreover, the overexpression of Snakin protein 1 in potato enhances resistance to relevant pathogens [46]. Additionally, Snakin-2 of French bean may be formed a 42 kDa protein complex with a proline-rich protein and participate in the plant defense process [47].Thus, in comparison with the reported about the orthologs of LsCysRP3 and 5, these L. styraciflua proteins may have antifungal activity. The expression pattern of their respective mRNA of LsCysRP3 and 5 during the interaction with N. parvum it is particularly interesting because of the repressive behavior profile of both mRNAs (Fig. 6). It is well known that many pathogens have evolved to generate tools with the aim of evading the host's immune system, so it will be interesting in future studies to investigate more about this effect.
Through genomic and transcriptomic analyses, has been already recognized that the pathogenic and virulence protein arsenal of N. parvum potentially encompass enzymes that facilitate woody degradation and host colonization [48,49]. In this current status of knowledge is insert our research with the aim to identify new cysteine rich proteins with potential roles in pathogenesis. After examine the genomic information of N. parvum, five sequences were selected with high cysteines content, the analysis identified the NpCysRP5 as a CFEM domain protein, distinguishable from other cysteine rich domains and inherent to fungi [37]. Even is already published that this class of motif is well represented in the Ascomycota phylum, the pioneers researches about CFEM domain [37,50] did not include members of Botryosphaeriaceae family. In this sense, NpCysRP5 was identified for first time as orthodox CFEM protein of N. parvum expressing early during infection process in Liquidambar.
Interestingly, NpCysRP4 (a protein apparently without a transmembrane domain) has a CFEM-like domain with two additional well-conserved cysteines (Fig. S6). The presence of these cysteines in NpCysRP4 could propitiate a disulfide rearrangement and conformation stability [51,52] and may increase the possibility of form more proteoforms with complementary functions contributing to virulence. In accordance with the reported by Zhang and collaborators a positive correlation between CFEM domain occurrence and fungal pathogenicity was showed [37] and it seems that occurrence of CFEM domain proteins is independently of the pathogen lifestyle (i.e. biotrophic, hemi biotrophic or necrotrophic). For example, in Botrytis cinerea (a necrotrophic fungi), BcCFEM1 is express highly at early stages of infection in Phaseolus vulgaris and the gene disruption results in decreased virulence [53]. For Magnaporthe grisea, a hemibiotrophic filamentous ascomycete, the mutant pth11 (a CFEM transmembrane protein) is impaired in the appressorium maturation in consequence in the infection capacity [54]. However, it is important to notice that not all CFEM proteins have a role in pathogenicity, for example three CFEM-motif GPI-anchored proteins from A. fumigatus participate in cell wall stability but not in fungal virulence [55]. Here, we identified a CFEM motif-containing protein of N. parvum (NpCysRP5) that also contain a putative transmembrane helix and its expressed during the early infection stages in L. styraciflua. It will be exciting to elucidate additional clues about it exactly contribution in pathogenesis. Finally, the phylogenetic analyses identified orthologs of NpCysRP4 and NpCysRP5 in more Botryosphaeriaceae species contributing to the knowledge of this important woody pathogens (Fig. 5). Additionally, the expression analysis indicated a contrasting mRNA expression profile of NpCysRP4 versus NpCysRP5 (Fig. 6), and because only mRNA NpCysRP5 expression showed a positive correlation during early times of infection, we thought that NpCysRP5 is a better candidate with a significant role during pathogenesis, emphasizing the fact that NpCysRP4, with a CFEM-like domain, have another important functions that may be discover in future works.
Besides of the new CFEM-like motif, NpCysRP2 also introduces a new cysteine rich domain with the enlisted characteristics: 1) the consensus motif deduced is C 1 [Y/F]xPx 9 − transmembrane helix towards the carboxyl terminal end is also conserved. These characteristics strongly suggest the existence of a new group of proteins and because an augmented expression was observed at early times, NpCysRP2 also is a good candidate to have a role during pathogenesis. All these data support the fact that NpCysRP2 is a new protein here we named as fungal cystine rich transmembrane protein (FCRTP).
Finally, phylogenetic analyses indicated two important deductions: 1) NpCysRPs are present in various fungal families and 2) each NpCysRPs are associated with particular species, for example for NpCysRP4 the order Eurotial is well represented with several Penicillium species while for NpCysRP5 the order Hypocrales is represented with members of Fusarium genus proposing that Neofusicocum parvum is as a very versatile fungus.

Conclusions
In this study, we documented that N. parvum is an aggressive pathogen of L. styraciflua. Besides, we identified novel CysRPs of both organisms as the NpCysRP2 that it seems to be conserved through

DNA extraction and molecular identification of Liqui 1-3 isolate by PCR
Each isolate was grown on PDA in 10 cm diameter Petri dishes for 9-10 days at 28ºC in darkness. The mycelium was collected with a scalpel and pulverized to isolate genomic DNA according to the protocol described by Tapia-Tussell et al., [56] with minor modifications. Specifically, in the lysis step we add 1 mL of SDS buffer, and the purification step was replaced with 500 µL phenol: chloroform: isoamyl alcohol (Sigma, Cat. P2069). For the molecular identification of Liqui 1-3 isolate we used primers already published that amplified regions of the e β-tubulin gene [57], the internal transcribed spacer (ITS) regions [58], a portion of RNA polymerase II subunit (RPB2) [59], the locus BotF15, an unknown locus containing microsatellite repeats [60] and the portion of gene encoding translation elongation factor 1 alfa (EF-1α) [59] see supplementary Table 1S. The five-region gene were amplified using the polymerase chain reaction (PCR) from genomic DNA using Platinum® Taq  The sizes and concentration of amplicons were visually checked on an electrophoresis 1% agarose gel and PCR products are purified using a Wizard SV gel and PCR clean-up system (Promega, ref A9281), and the DNA concentration was measured on a NanoDrop 2000c spectrophotometer (Thermo scientific). Samples were sent to Langebio, Cinvestav, for sequencing. DNA sequences were analyzed using nucleotide collection (nr/nt) optimize for highly similar sequences (megablast) in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Pathogenicity assay in Arabidopsis thaliana seedlings Seeds of Arabidopsis thaliana ecotype Col-0 were surface-disinfected with 96% (v/v) ethanol for 7 min and 20% (v/v) bleach for 7 min, then rinsed five times for 5 min with distilled water and stratified for 2 days at 4 ºC. Seeds were grown on each agar plates (10 seeds per plate) containing 0.2X Murashige and Skoog (1962) medium (MS basal salts mixture, Phytotechnology Laboratories, Cat. M524),0.6% sucrose (Sucrose: Fluka Analytical, Cat. 84100) and 1% Agar Plant TC at pH 7. Plates were incubated at 21 ± 1 ºC in a 12-h-light/12-h-dark cycle for 7 days. After this time, the seedlings were inoculated with 0.5 cm x 0.5 cm agar plug containing the isolate. Then, the plates were incubated for seven additional days in the same previous conditions and the photograph was taken with a Nikon camera D3200. All the experiments were carried out in triplicates.

Pathogenicity assay in Liquidambar styraciflua leaves and stems
Liquidambar styraciflua healthy leaves and stems were collected from the cloud forest Santuario del Bosque de Niebla at Xalapa, Veracruz in Mexico. Once they were collected, they were immediately placed in plastic bags that contained sterile water to conserve humidity. The leaves were collected and sterilized the same day that the experiment was carried out. The leaves were sterilized with 2% sodium hypochlorite for 1 minute, then washed five times with sterile distilled water. Three sterilized leaves were placed in a humid chamber. The humid chambers were prepared by placing a circle of sterile filter paper in the bottom of a Petri dish (150 × 20 mm) previously sterilized with UV light for 15 min. Approximately 4-5 mL of sterile distilled water was added to filter paper. The leaves (3) were mechanically damaged with a sterile scalpel in the base and then inoculated with a plug of approximately 0.5 × 0.5 cm of N. parvum (Liqui 1-3) previously grown on PDA medium. Leaves with the damage in the base of the leaf and a control with damage in the base of the leaf were used as a control. Finally, the control and inoculated leaves were incubated at 25 ± 2 ˚C with 80% of relative humidity in darkness in a plant growth chamber (Thermo Scientific, 3768). The photographs were taken at 0, 1, 3, 8 and 18 dpi with a Nikon camera D3200.
Stem infection experiments were carried out as followed: previously sterilized young branches of adult tree of L. styraciflua were cut with a scalpel of a single-edged knife into fragments of approximately 10 cm and then were cut longitudinally. The L. styraciflua stems were placed upward by quadruplicate inside a humid chamber that were prepared as previously described. The plant stems were infected on the center of the vascular tissue with a plug of approximately 0.5 × 0.5 cm of N. parvum (LSH1-083) previously grown on PDA medium. We included a negative control of a PDA plug without fungus. Finally, it was kept in darkness and incubated at 25 ± 2 ˚C with 80% of relative humidity in a plant growth chamber (Thermo Scientific, 3768).

Phylogenetic Trees
Phylogenetic analysis were construct with Mega X software [68] using the Maximum Likelihood method and JTT matrix-based model [69], Jalview software was used for perform the alignments [70], with Tcoffee method [71], and finally BLAST tool was used to find similarity of sequence in other fungus.

Rna Extraction And Gene Expression
Total RNA was extracted using the Plant/Fungi Total RNA Purification Kit (Norgen Bioteck) following the manufacturers recommendations. The amount of RNA was determined with f a spectrophotometer (NanoDrop™ 2000c; Thermo Scientific™) and its integrity was evaluated with the ratio Oligo specificity was followed by a melting curve analysis with continuous fluorescence data acquisition during the 95 − 55 °C melt. For oligos design the primer3 software was used followed the parameters suggest by Thornton and Basu [72]. Relative expression levels for validated genes were calculated by the method [73]and oligos for Tip41, actin and ubiquitin, of Liquidambar styraciflua were evaluated as reference gene (all primers are listed in Supplementary Table S1). The geometric mean between Tip41 and actin was used as a reference gene [74].       Expression at early times of infection of CysRPs mRNAs of both L. styraciflua and N. parvum.
A. LsCysRPs mRNA expression at 1 and 3 dpi. For the analysis the geometric mean between Tip41 and actin was used as a reference gene. For all the transcripts statistic significantly, difference was detected between 1 and 3 dpi at P < 0.01, using Student's t test. B.
NpCysRPs mRNAs expression from 1 to 3 dpi. NpCysRP3 was used as a reference gene. Data are the average of three independent inoculation experiments.

Supplementary Files
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