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 H2O2 (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. Meanwhile in N. parvum 6, 4, 4, 5 and 4 disulfide bridges were estimated for NpCysRP1, NpCysRP2, 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 was conducted (Figs. 4 and 5). Clearly, all the NpCysRPs were group in the Botryopshaeria lineage including Lasiodiplodia, Diplodia and Macrophomina species, however the species in the subclade not always were the same, NpCysRP1, 2 and 5 were better related to Lasiodiplodia theobromae while NpCysRP3 and 4 shared the branch with Macrophomina phaseolina.
NpCysRP1 phylogeny revealed the existence of few orthologs sequences for this protein in databases and occurred in the Botryosphaeriaceae family only in the species L. theobromae (85.00% identity), D. corticola (85.42% identity) and D. seriata (82.29%, identity), as well as in family Cordycipitaeceae, order Hypocreales, characterized by containing entomopathogenic fungi species as Beauveria bassiana and Cordyceps confragosa but with low identities (29.67% and 31.36% respectively) 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 C1[Y/F]xPx9 − 10C2x6−8C3C4x4C5x2Nx2C6x10 − 23C7Tx8C9x3C10 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. theobromae and D. corticola presented a transmembrane domain crossing at carboxyl-end. Moreover, the predicted proteoforms for all the sequences used in the phylogenetic analysis (26 in total) displayed the transmembrane helix (Fig. S6).
A particular phylogeny was noted in the case of NpCysRP3 (Fig. 4C), this protein not present a well-defined clade distribution within the Botryosphaeriaceae family and only Macrophomina phaseolina orthologs (MpCysRP A, B and C) were found with 64.46, 29.17 and 30.83% identity. Interesting, Nactriaceae family was represented in the phylogenic tree with some members of the unique and fascinating Ambrosia fusarium clade represented by Fusarium euwallaceae, and Fusarium kuroshium that has been recognized recently as an emerging fungal pathogen [35, 36].
NpCysRP5 has a CFEM domain characterized by containing eight cysteines with the particular consensus motif PxC1[A/G]x2C2x8-12C3x1−3[x/T]Dx2 − 5C4xC5x9 − 14C6x3−4C7x15 − 16C8, [37]. Interestingly, NpCysRP4 and orthologs have a CFEM-like domain with conserved extra cysteines pair (C58 and C94 in 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 share the subclade with M. 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. For LsCysRP3 the BLAST® result reveled a sequence identity of 66.98–64.15% with various Gossypium species and 63.81% with Vitis pseodoreticulata. Finally, LsCysRP5 showed 73.83–65.42% identity with different Quercus species, 74.77% Castanea mollissima and 67.29% with Durio zibethinus.
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 Fig. 6A is showed the LsPR1 expression levels, an orthologous of Nicotiana tabacum gene encoding the pathogenesis-related protein 1 (PR1), a protein involved in defense response in plants and usually used as a defense marker, qPCR results showed an increase of LsPR1 mRNA at 1 and 3 dpi. An opposite profile was observed in all LsCysRPs transcripts, because the expression decreased after 1 dpi, LsCysRP2 presented the lowest level at this time post infection. At 3 dpi, LsCysRP2 presented the most significant changed and LsCysRP3 remained unchanged, while LsCysRP1, 4 and 5 presented a mild increase.
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.