Morphological and transcriptional analysis of <i>Colletotrichum lindemuthianum</i> race 7 during early stages of infection in common bean

Romero, German; González, Sandra; Royero, Wendy; González, Adriana

doi:10.1590/1678-4685-GMB-2022-0263

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Genetics and Molecular Biology

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Genetics of Microorganisms • Genet. Mol. Biol. 47 (1) • 2024 • https://doi.org/10.1590/1678-4685-GMB-2022-0263 copy

Morphological and transcriptional analysis of Colletotrichum lindemuthianum race 7 during early stages of infection in common bean

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

The infection process of the hemibiotrophic fungus Colletotrichum lindemuthianum has been independently studied at the microscopic and genomic levels. However, the relationship between the morphological changes and the pathogenicity mechanisms of the fungus at the early stages of the infection remains uncharacterized. Therefore, this study attempts to bridge this gap by integrating microscopic and transcriptional approaches to understand the infection process of C. lindemuthianum. Fungal structures were followed by fluorescence microscopy for 120 hours. Simultaneously, the transcriptomic profile was made using RNAseq. Morphological characterization shows that appressoria, infective vesicles, and secondary hypha formation occur before 72 hours. Additionally, we assembled 38,206 transcripts with lengths between 201 and 3,548 bp. The secretome annotation revealed the expression of 1,204 CAZymes, of which 17 exhibited secretion domains and were identified as chitinases and β-1,3-glucanases, 27 were effector candidates, and 30 were transport proteins mostly associated with ABC-type. Finally, we confirmed the presence and expression of CAC1 role during the appressoria formation of Clr7. This result represents the first report of adenylate cyclase expression evaluated under three different approaches. In conclusion, C. lindemuthianum colonizes the host through different infection structures complemented with the expression of multiple enzymes, where CAC1 favors disease development.

Keywords:
Anthracnose; RNAseq; CAZymes; Transporter protein; effector protein

Time (Hours)	Gene name	% ID	e-value	Protein annotation	Uniprot Accesion	Reported fungal pathogen	References
24	CBP1	64,51	1.16e-05	Chitin deacetylase	G4N4F9	Magnaporthe oryzae	Dean et al., 2005Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986.
24	bcsod1	56,20	9.82e-54	Superoxide dismutase	Q70Q35	Botrytis cinerea	Rolke et al., 2004Rolke Y, Liu S, Quidde T, Williamson B, Schouten A, Weltring KM, Siewers V, Tenberge KB, Tudzynski B and Tudzynski P (2004) Functional analysis of H(2)O(2)-generating systems in Botrytis cinerea: The major Cu-Zn-superoxide dismutase (BCSOD1) contributes to virulence on French bean, whereas a glucose oxidase (BCGOD1) is dispensable. Mol Plant Pathol 5:17-27.
24	CBP1	80,00	5.06e-05	Chitin deacetylase	G4N4F9	Magnaporthe oryzae	Dean et al., 2005Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986.
24	MoPtc2	43,13	6.53e-06	Protein phosphatase 2C	Q4WTH5	Magnaporthe oryzae	Nierman et al., 2005Nierman WC, Pain A, Anderson MJ, Wortman JR, Kim HS, Arroyo J, Berriman M, Abe K, Archer DB, Bermejo C et al. (2005) Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438:1151-1156.
24	CBP1	81,81	3.10e-06	Chitin deacetylase	G4N4F9	Magnaporthe oryzae	Dean et al., 2005Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986.
24	MGG_10510	56,61	4.53e-10	Ribonuclease T2	G4MS40	Magnaporthe oryzae	Dean et al., 2005Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986.
24	mac1	33,33	7.50e-12	Adenylate cyclase	O13328	Magnaporthe oryzae	Dean et al., 2005Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986.
48	bcsod1	55,92	3.54e-50	Superoxide dismutase	Q70Q35	Botrytis cinerea	Rolke et al., 2004Rolke Y, Liu S, Quidde T, Williamson B, Schouten A, Weltring KM, Siewers V, Tenberge KB, Tudzynski B and Tudzynski P (2004) Functional analysis of H(2)O(2)-generating systems in Botrytis cinerea: The major Cu-Zn-superoxide dismutase (BCSOD1) contributes to virulence on French bean, whereas a glucose oxidase (BCGOD1) is dispensable. Mol Plant Pathol 5:17-27.
48	FZC9	33,71	1.37e-26	Transcriptional activator xlnR	G5EH28	Magnaporthe oryzae	Dean et al., 2005Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986.
48	CBP1	64,52	1.27e-05	Chitin deacetylase	G4N4F9	Magnaporthe oryzae	Dean et al., 2005Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986.
48	CRU1	38,48	7.43e-06	Cell cycle regulatory protein	Q8J214	Ustilago maydis	Unpublished
48	mac1	40,23	1.42e-09	Adenylate cyclase	O13328	Magnaporthe oryzae	Choi and Dean, 1997Choi W and Dean R (1997) The Adenylate Cyclase Gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development. Plant Cell 9:1973-1983.
48	CBP1	81,82	2.70e-06	Chitin deacetylase	G4N4F9	Magnaporthe oryzae	Dean et al., 2005Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986.
48	PDIG_23520	57,34	4.72e-47	Cysteine-rich Anionic (Sca) protein	K9G4Z7	Penicillium digitatum	Marcet-Houben et al., 2012Marcet-Houben M, Ballester AR, de la Fuente B, Harries E, Marcos JF, González-Candelas L and Gabaldón T (2012) Genome sequence of the necrotrophic fungus Penicillium digitatum, the main postharvest pathogen of citrus. BMC Genomics 13:646.
48	CBP1	80,00	1.15e-04	Chitin deacetylase	G4N4F9	Magnaporthe oryzae	Dean et al., 2005Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986.
48	GzGPB1	53,02	1.19e-83	G protein beta subunit	I1RJS2	Fusarium graminearum	Cuomo et al., 2007Cuomo CA, Güldener U, Xu JR, Trail F, Turgeon BG, Di Pietro A, Walton JD, Ma LJ, Baker SE, Rep M et al. (2007) The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science 317:1400-1402.
48	Pstg_13661	45,30	1.01e-47	chitin deacetylase	A0A0L0V0Y3	Puccinia striiformis	Duplessis et al., 2011Duplessis S, Cuomo CA, Lin YC, Aerts A, Tisserant E, Veneault-Fourrey C, Joly DL, Hacquard S, Amselem J, Cantarel BL et al. (2011) Obligate biotrophy features unraveled by the genomic analysis of rust fungi. Proc Natl Acad Sci U S A 108:9166-9171.
48	UvHrip1	69,52	5.73e-53	Effector protein	A0A1B5KZM5	Ustilaginoidea virens	Kumagai et al., 2016Kumagai T, Ishii T, Terai G, Umemura M, Machida M and Asai K (2016) genome sequence of Ustilaginoidea virens IPU010, a rice pathogenic fungus causing false smut. Genome Announc 4:e00306-16.
72	CBP1	81,82	2.34e-06	Chitin deacetylase	G4N4F9	Magnaporthe oryzae	Dean et al., 2005Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986.
72	CBP1	41,07	3.99e-05	Chitin deacetylase	G4N4F9	Magnaporthe oryzae	Dean et al., 2005Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986.
72	Ubc9	38,00	9.09e-05	SUMO-conjugating enzyme ubc9	G4ND54	Magnaporthe oryzae	Dean et al., 2005Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986.
72	DCL1	39,01	5.07e-24	Dicer-like protein 1	A0A194VW85	Valsa mali	Yin et al., 2015Yin Z, Liu H, Li Z, Ke X, Dou D, Gao X, Song N, Dai Q, Wu Y, Xu JR et al. (2015) Genome sequence of Valsa canker pathogens uncovers a potential adaptation of colonization of woody bark. New Phytol 208:1202-1216.
72	Mouba2	51,13	1.44e-93	Ubiquitin-activating enzyme E1-like	G4MLK2	Magnaporthe oryzae	Dean et al., 2005Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986.
72	Pstg_13661	47,74	1.11e-45	chitin deacetylase	A0A0L0V0Y3	Puccinia striiformis	Duplessis et al., 2011Duplessis S, Cuomo CA, Lin YC, Aerts A, Tisserant E, Veneault-Fourrey C, Joly DL, Hacquard S, Amselem J, Cantarel BL et al. (2011) Obligate biotrophy features unraveled by the genomic analysis of rust fungi. Proc Natl Acad Sci U S A 108:9166-9171.
72	LmABCB3	36,13	2.69e-17	MDR efflux pump ABC3	Q3Y5V5	Magnaporthe oryzae	Dean et al., 2005Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986.
72	bcsod1	56,21	9.82e-54	Superoxide dismutase	Q70Q35	Botrytis cinerea	Rolke et al., 2004Rolke Y, Liu S, Quidde T, Williamson B, Schouten A, Weltring KM, Siewers V, Tenberge KB, Tudzynski B and Tudzynski P (2004) Functional analysis of H(2)O(2)-generating systems in Botrytis cinerea: The major Cu-Zn-superoxide dismutase (BCSOD1) contributes to virulence on French bean, whereas a glucose oxidase (BCGOD1) is dispensable. Mol Plant Pathol 5:17-27.
72	MGG_10510	26,71	3.44e-10	Ribonuclease T2	G4MS40	Magnaporthe oryzae	Dean et al., 2005Dean RA, Talbot NJ, Ebbole DJ, Farman ML, Mitchell TK, Orbach MJ, Thon M, Kulkarni R, Xu JR, Pan H et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434:980-986.

Hours after inoculation (hai)	CAZyme Family	Total	Enzyme description
24	GH3	2	Xylan 1,4-β-xylosidase
24	GH18	1	Chitinase/lysozyme
24	GH19	4	Chitinase
24	GH152	9	β-1,3-glucanase
48	CE4	1	Chitin deacetylase
48	GH3	1	Xylan 1,4-β-xylosidase
48	GH16	1	Xyloglucan:xyloglucosyltransferase
48	GH18	1	Chitinase/lysozyme
48	GH19	4	Chitinase
48	GH152	7	β-1,3-glucanase
72	CE4	1	Chitin deacetylase
72	GH1	1	β-glucosidase
72	GH3	1	Xylan 1,4-β-xylosidase
72	GH18	1	Chitinase/lysozyme
72	GH19	1	Chitinase
72	GH27	1	α-galactosidase
72	GH152	7	β-1,3-glucanase

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[1] Send correspondence to
Adriana González-Almario. Universidad Nacional de Colombia, Facultad de Ciencias Agrarias, Carrera 30 No. 45-03, Bogotá, Colombia. E-mail: adgonzalezal@unal.edu.co.