Abstract
Effectors are secreted by plant-associated microorganisms to modify the host cell physiology. As effectors, the Necrosis- and Ethylene-inducing peptide 1-like proteins (NLPs) are involded in the early phases of plant infection and may trigger host immune responses. Corynespora cassiicola is a polyphagous plant pathogen that causes target spot on many agriculturally important crops. Using genome assembly, gene prediction, and proteome annotation tools, we retrieved 135 NLP-encoding genes from proteomes of 44 isolates. We explored the evolutionary history of NLPs using Bayesian phylogeny, gene genealogies, and selection analyses. We accessed the expression profiles of the NLP genes during the early phase of C. cassiicola–soybean interaction. Three NLP putative-effector genes (Cc_NLP1.1, Cc_NLP1.2A, and Cc_NLP1.2B) were maintained in the genomes of all isolates tested. An NLP putative-non-effector gene (Cc_NLP1.3) was found in three isolates that had been originally obtained from soybean. Putative-effector NLPs were under different selective constraints: Cc_NLP1.1 was under stronger selective pressure, while Cc_NLP1.2A was under a more relaxed constraint. Meanwhile, Cc_NLP1.2B likely evolved under either positive or balancing selection. Despite highly divergent, the putative-effector NLPs maintain conserved the residues necessary to trigger plant immune responses, suggesting they are potentially functional. Only the Cc_NLP1.1 putative-effector gene was significantly expressed at the early hours of soybean colonization, while Cc_NLP1.2A and Cc_NLP1.2B showed much lower levels of gene expression.
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The authors confirm that the data supporting the findings of this study are available in the supplementary material. Additional data are available from the corresponding author upon request.
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Acknowledgements
We thank Dr. Eveline Caixeta and the Laboratório de Biotecnologia do Cafeeiro for the use of the Real-Time PCR System. This work was supported by The Minas Gerais State Foundation of Research Aid—FAPEMIG (grant number APQ-00150-17) and by The National Council of Scientific and Technological Development – CNPq (fellowship number PQ 302336/2019-2) to LOO. TCSD received student fellowships from the CAPES Foundation (PROEX—0487 No. 1684083) and CNPq (GM/GD 142400/2018-1).
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TCSD performed data assembly and analyses. TCSD, HVSR, and LOO contributed to the study conception. VDR and MDLC contributed to the gene expression analyses. TCSD and LOO wrote the manuscript. LOO and HVSR supervised the research. All authors read and approved the final manuscript.
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294_2022_1252_MOESM1_ESM.pdf
Fig. S1. Sliding window neutrality tests of three predicted Necrosis- and Ethylene-inducing peptide 1-like protein (NLP)-encoding genes (CDS) of Corynespora cassiicola. The x axis shows the midpoints of contiguous windows (25 bases, with steps of 5 bases). The gray vertical bar indicates the relative position of the GHRHDWE motif. The arrow indicates sites with significant departures from zero (p-value < 0.05)
294_2022_1252_MOESM2_ESM.pdf
Fig. S2. Placement of nucleotide substitutions along the haplotypes of each of the putative-effector three NLP-encoding genes (Cc_NLP1.1, Cc_NLP1.2A, and Cc_NLP1.2B) (CDS) in Corynespora cassiicola. The putative-effector NLP-encoding genes from isolate CC_29 were used as reference for haplotype sequence alignment, according to the haplogroups in Fig. 2. Upper protein representations indicate portions of the CDS that encode for the signal peptide (SP), the NPP1 domain, and the conserved GHRHDWE motif in the putative-effector NLPs of isolate CC_29. A. Cc_NLP1.1 compared to CC_29_g12228 (haplotype 3). B. Cc_NLP1.2A compared to CC_29_g13117 (haplotype 15). C. Cc_NLP1.2B compared to CC_29_g13473 (haplotype 8). Plus (+) signs indicate substitutions that resulted in amino acids replacements predicted to be positively selected, according to codeml/PAML analysis. Types of nucleotide substitutions as indicated
294_2022_1252_MOESM3_ESM.pdf
Fig. S3. Expression patterns of the Necrosis- and Ethylene-inducing peptide 1-like (NLP) genes of Corynespora cassiicola (isolate CC_29) after inoculation in soybean plants. Gene expression was inferred by RT-qPCR analyses. The time (x axis) is given in hours post inoculation (hpi). The fold variation of gene expression of each NLP gene (y axis) was calculated using the 2-ΔCtmethod, the values were normalized to an endogenous control gene (β-tubulin). Letters show significant differences (p-value < 0.05) after a one-way ANOVA followed by Tukey’s test for each NLP gene individually. Error bars represent the standard deviation of three biological replicates
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Dal’Sasso, T.C.d., Rocha, V.D., Rody, H.V.S. et al. The necrosis- and ethylene-inducing peptide 1-like protein (NLP) gene family of the plant pathogen Corynespora cassiicola. Curr Genet 68, 645–659 (2022). https://doi.org/10.1007/s00294-022-01252-0
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DOI: https://doi.org/10.1007/s00294-022-01252-0