Identification of N. parvum as a pathogen of L. styraciflua
Eleven fungi were isolated from L. styraciflua leaves with visible symptoms such as necrosis and discoloration. The potential pathogenicity of the fungi was tested in L. styraciflua leaves and in seedlings of the plant model Arabidopsis thaliana (Fig. 1 and Additional file 1: Fig. S1). The pathogenicity screening identified the fungal Liqui 1-3 strain as the most pathogenic, since in A. thaliana seedlings, the fungus covered all the plant tissues, provoking severe leaf discoloration at 7 days post inoculation (dpi). L. styraciflua leaves developed clear necrosis and discoloration, including at 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 critical pathogenic effect; for example, while Liqui 2-2 covered more than 50% of the seedlings, the foliar tissue of the infected plants showed greater vigor than that of the control. Liqui 1-2-01, Liqui 1-2-03 and Liqui 3-2 at 7 dpi showed a discrete pathogenic effect. A. thaliana developed a shorter primary root but an increased number of secondary roots during the interaction with these isolates. The isolates Liqui 1-04, Liqui 1-01, Liqui 3-3 and Liqui 3-1 had no effect in L. styraciflua leaves or A. thaliana seedlings.
To identify the Liqui 1-3 strain at the molecular level, universal and specific primers were used (Additional file 2: Fig. S2 and Additional file 3: Table S1). The analysis revealed that Liqui 1-3 belongs to the Botryosphaeriaceae family as a member of the species N. parvum.
Establishment of the L. styraciflua–N. parvum pathosystem
Because we were interested in studying the interaction between L. styraciflua and N. parvum, we established a 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 over time (Fig. 2), and there was distinct petiole necrosis at 8 and 16 dpi; the presence of a whitish mycelium in the necrotic leaf area was also evident. To characterize the infection process in more detail, we analyzed the infected and uninfected tissue using SEM (Fig. 3). SEM images revealed that the fungus grew robustly on the leaf (adaxial) surface, forming a hyphal mass and causing tissue degradation; the cuticle and wax integrity were compromised (Fig. 3a-d). In addition, the infection provoked petiole degradation. A transverse cut of the leaf base showed that the fungus was able to develop pycnidia, an asexual reproductive structure. The pycnidia appeared individually or as aggregates embedded in the plant tissue with thick walls composed of numerous cells (Fig. 3i-l). A longitudinal section of pycnidium showed mature conidia. The conidiogenic cells without septa and with an oval shape were localized perpendicular to the walls of the pycnidium. Because the Botryosphaeriaceae family members are characterized as woody-plant pathogens, we tested the pathogenicity of the Liqui 1-3 strain in fresh stems of L. styraciflua. As shown, N. parvum at 7 dpi triggered distinct symptoms of disease, such as discoloration and necrosis, that covered a zone beyond the site of inoculation (Additional file 4: Fig. S3).
Detection of hydrogen peroxide in L. styraciflua leaves at early stages of the infection process
Reactive oxygen species (ROS) accumulate in plant cells during pathogen infection and may cause oxidative damage to proteins, DNA, and lipids or act as signaling molecules to regulate the defense response [33, 34]. One such species is hydrogen peroxide, and by diaminobenzidine (DAB) staining, we clearly detected the presence of a dark brown precipitate in the infected leaves at early time points (1 and 3 dpi), indicating the presence of H2O2 (Additional file 5: Fig. S4).
Identification of CysRPs in L. styraciflua and N. parvum and their general features
CysRPs have been widely studied for their important functions in plant-pathogen interactions, and to identify CysRPs in L. styraciflua and N. parvum, two databases with transcriptomic and genomic information were analyzed (see Materials and Methods). For each organism, five sequences encoding CysRPs were identified (LsCysRP1-5 and NpCysRP1-5), all of which contained a putative start and stop codon with the exception of the LsCysRP3 sequence, in which a stop codon was not present (Table 1 and Additional file 6: Table S2). The amino acid sequence length varied between 95 and 204, and the molecular weight ranged between 7.7 and 17.5 kDa. In addition, the cysteine content was determined. Interestingly, with the exception of LsCysRP1, which had a calculated isoelectric point (pI) of 6.04, the calculated pI values of all the LsCysRPs were greater than 8.67, while, with the exception of NpCysRP1, which had a pI of 7.57, the calculated pI values of all the NpCysRPs were less than 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, and LsCysRP5 have the potential to form 3, 4, 4, 0, and 6 disulfide bonds, respectively. Meanwhile, in N. parvum, 6, 4, 4, 5 and 4 disulfide bridges were estimated for NpCysRP1, NpCysRP2, NpCysRP3, and NpCysRP4 NpCysRP5, respectively. A clear discrepancy regarding the identification of the cysteines that form the pairs was detected among the methods (Additional file 7: Table S3).
To determine whether the CysRPs have the potential to be secreted, an analysis with the TargerP-2.0 server was conducted. All sequences with the exception of the LysCysRP2 sequence have a peptide signal between 17 and 27 amino acids in length. To corroborate these results, additional analyses were performed using the Protter and DeepLoc-1.0 servers, and the results indicated that all the CysRPs have a signal peptide and are extracellular proteins (Table 2).
To identify possible functions and regions of similarity in CysRPs, BLAST and MOTIF tools were used. No significant similarity was found for LsCysRP1, 2 and 4 or for NpCysRP1, 2 and 3. In contrast, LsCysRP3 showed similarity with an LTP and LsCysRP5 with a gibberellin-regulated protein 1-like protein. Interestingly, for both NpCysRP4 and 5, a CFEM domain was identified (Table 1, Fig. 6S).
CysRP phylogenetic analyses
To obtain more information about NpCysRPs, a corresponding phylogenetic analysis for each of these proteins was conducted (Fig. 4 and 5). Clearly, all the NpCysRPs were grouped in the Botryopshaeria lineage, including Lasiodiplodia, Diplodia and Macrophomina species; however, the species in the subclade were not always the same. NpCysRP1, 2 and 5 were closely related to Lasiodiplodia theobromae, while NpCysRP3 and 4 shared a branch with Macrophomina phaseolina.
The NpCysRP1 phylogeny revealed the existence of few orthologous sequences for this protein in databases, and this protein occurred in the Botryosphaeriaceae family only in the species L. theobromae (85.00% identity), Diplodia corticola (85.42% identity) and Diplodia seriata (82.29%, identity), as well as in the family Cordycipitaeceae, order Hypocreales, characterized by entomopathogenic fungal species such as Beauveria bassiana and Cordyceps confragosa but with low identities (29.67% and 31.36%, respectively); however, the cysteines remained at the same site in the sequences (Additional file 8: Fig. S5). Other species were included in the alignment, such as Aspergillus leporis and Rhizoctonia solani; however, these sequences have a lower number of cysteines and share lower identity.
In addition to the Botryosphaeria lineage, the NpCysRP2 phylogenetic tree contained orthologs of the family Hypocreaceae, order Hypocreales, including seven different species of the Trichoderma genus with identities that ranged from 33.04 to 38.18%. Additionally, members of the family Glomerellaceae were included, such as species of the well-known phytopathogenic genus Colletotrichum, such as the species Colletotrichum asianum, Colletotrichum nymphaeae and Colletotrichum orchidophillum, with 33.04, 36.28 and 37.19% amino acid identity, respectively. Interestingly, the tree also showed a species of the order Sordariales, the human pathogen Madurella mycetomatis, with 33.90% identity. The alignment revealed that NpCysRP2 introduces a new cysteine-rich domain with the consensus motif C1[Y/F]xPx9-10C2x6-8C3C4x4C5x2Nx2C6x10-23C7Tx8C9x3C10 at the N-terminus. Additionally, the multiple-sequence 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 acids, respectively (Additional file 8: Fig. S5). Interestingly, the proteoforms of L. theobromae and D. corticola presented a transmembrane domain crossing at the carboxyl-end. Moreover, the predicted proteoforms for all the sequences used in the phylogenetic analysis (26 in total) showed the transmembrane helix (Additional file 9: Fig. S6).
A particular phylogeny was noted in the case of NpCysRP3 (Fig. 4C); this protein did not present a well-defined clade distribution within the Botryosphaeriaceae family, and only the M. phaseolina orthologs (MpCysRPs A, B and C) were found, with 64.46, 29.17 and 30.83% identity, respectively. Interestingly, the Nactriaceae family was represented in the phylogenetic tree with some members of the unique and fascinating Ambrosia Fusarium clade represented by Fusarium euwallaceae and by Fusarium kuroshium, which has been recognized recently as an emerging fungal pathogen [35, 36].
NpCysRP5 has a CFEM domain characterized by eight cysteines with the specific consensus motif PxC1[A/G]x2C2x8-12C3x1-3[x/T]Dx2-5C4xC5x9-14C6x3-4C7x15-16C8 . Interestingly, NpCysRP4 and its orthologs have a CFEM-like domain with a conserved extra cysteine pair (C58 and C94 in the N. parvum sequence) forming the consensus motif PxC1[A/G]x2C2x8-12C3x1-3[x/T]Dx2-5C4xC5x8-13C6C7x3-4C8x15-16C9x12-13C10. NpCysRP4 and NpCysRP5 are well represented in the clade of the Botryosphaeriaceae family, but NpCysRP4 shares a subclade with M. phaseolina, with 78.07% identity, while NpCysRP5 showed 83.01% identity with L. theobromae. Cenococcum geophilum and Glonium stellatum belong to the Gloniaceae family and present orthologs for both NpCysRP4 and NpCysRP5. Interestingly, for NpCysRP4, various orthologs in the Nectriaceae family were identified, including in the order Hyporcreales represented by the well-known phytopathogenic Fusarium species Fusarium verticillioides and Fusarium oxysporum, which were not found in the NpCysRP5 phylogenetic tree using 33 sequences. In the case of NpCysRP5, a clear clade represented by the order Eurotiales was identified, in which different Penicillium species were grouped, 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 not possible to construct the corresponding phylogenetic trees. For LsCysRP3, the BLAST® result revealed a sequence identity of 66.98-64.15% with various Gossypium species and 63.81% with Vitis pseudoreticulata. Finally, LsCysRP5 showed 73.83-65.42% identity with different Quercus species, 74.77% identity with Castanea mollissima and 67.29% identity with Durio zibethinus.
Expression of CysRP mRNAs of L. styraciflua and N. parvum during early times of the infection process.
To explore whether transcription of CysRP mRNAs of L. styraciflua and N. parvum was modulated at early stages of the interaction, quantitative polymerase chain reaction (qPCR) tests were performed. Fig. 6A shows the expression levels of LsPR1, an ortholog of the Nicotiana tabacum gene encoding pathogenesis-related protein 1 (PR1), a protein involved in the defense response in plants and usually used as a defense marker. The qPCR results showed an increase in LsPR1 mRNA at 1 and 3 dpi. The opposite profile was observed for all LsCysRP transcripts, as the expression decreased after 1 dpi, and LsCysRP2 presented the lowest level at this time post infection. At 3 dpi, LsCysRP2 presented the most significant change, and LsCysRP3 remained unchanged, while LsCysRP1, 4 and 5 presented a mild increase.
Finally, our experimental design allowed us to compare the expression of NpCysRP transcripts between 1 and 3 dpi. Fig. 6B shows that there was an increase in the expression of NpCysRP1, 2 and 5 mRNAs, with NpCysRP5 showing the most significant increase. In contrast, NpCysRP4 showed a significant decrease.