The C2H2 Transcription Factor Con7 Regulates Vegetative Growth, Cell Wall Integrity, Oxidative Stress, Asexual Sporulation, Appressorium and Hyphopodium Formation, and Pathogenicity in Colletotrichum graminicola and Colletotrichum siamense

Abstract

The Colletotrichum genus is listed as one of the top 10 important plant pathogens, causing significant economic losses worldwide. The C₂H₂ zinc finger protein serves as a crucial transcription factor regulating growth and development in fungi. In this study, we identified two C₂H₂ transcription factors, CgrCon7 and CsCon7, in Colletotrichum graminicola and Colletotrichum siamense, as the orthologs of Con7p in Magnaporthe oryzae. Both CgrCon7 and CsCon7 have a typical C₂H₂ zinc finger domain and exhibit visible nuclear localization. Disrupting Cgrcon7 or Cscon7 led to a decreased growth rate, changes in cell wall integrity, and low tolerance to H₂O₂. Moreover, the deletion of Cgrcon7 or Cscon7 dramatically decreased conidial production, and their knockout mutants also lost the ability to produce appressoria and hyphopodia. Pathogenicity assays displayed that deleting Cgrcon7 or Cscon7 resulted in a complete loss of virulence. Transcriptome analysis showed that CgrCon7 and CsCon7 were involved in regulating many genes related to ROS detoxification, chitin synthesis, and cell wall degradation, etc. In conclusion, CgrCon7 and CsCon7 act as master transcription factors coordinating vegetative growth, oxidative stress response, cell wall integrity, asexual sporulation, appressorium formation, and pathogenicity in C. graminicola and C. siamense.

Keywords: C2H2 transcription factors; Colletotrichum graminicola; Colletotrichum siamense; appressorium formation; conidiation.

Figure 1

Bioinformatic analyses of CgrCon7 and CsCon7. (A) Protein domain analyses of CgrCon7 and CsCon7. The magenta box represents a low-complexity region. The green box represents a coiled-coil region. (B) Comparative alignment between deduced Colletotrichum Con7 protein sequences and orthologs from other plant pathogens. The deduced Con7 proteins were predicted to share a conserved C₂H₂ zinc finger domain, a nuclear localization signal (NLS), and a PEST motif for protein degradation. Cgr: Colletotrichum graminicola (XP_008089279.1), Cs: Colletotrichum siamense (XP_036502431.1), Mo: Magnaporthe oryzae (XP_003712849.1), Fg: Fusarium graminearum (XP_011321497.1).

Figure 2

Subcellular localization of CgrCon7 and CsCon7 in the conidium, appressorium, and hyphae. (A) Subcellular localization of CgrCon7 in C. graminicola. (B) Subcellular localization of CsCon7 in C. siamense.

Figure 3

Vegetative growth on four media. (A) Growth comparison of C. graminicola strains on four media. (B) Statistical analyses of colony diameters of C. graminicola strains. (C) Growth comparison of C. siamense strains on four media. (D) Statistical analyses of colony diameters of C. siamense strains. ** Significant at p < 0.01.

Figure 4

Effects of Con7 on the cell wall integrity of C. graminicola and C. siamense. (A) Effects of SDS and congo red (CR) on the growth of C. graminicola strains. (B) Statistical analyses of inhibition rates of C. graminicola strains. (C) Effects of SDS and CR on the growth of C. siamense strains. (D) Statistical analyses of inhibition rates of C. siamense strains. * Significant at p < 0.05; ** significant at p < 0.01. (E) CFW dyeing of the mycelia and falcate conidia of C. graminicola strains. s: septa; ch: chitin.

Figure 5

H₂O₂-sensitive assays. (A) Effects of H₂O₂ on the growth of C. graminicola strains on 3 dpi. (B) Effects of H₂O₂ on the growth of C. siamense strains on 2 dpi. (C) Statistical analyses of inhibition zone diameters of C. graminicola strains. (D) Statistical analyses of inhibition zone diameters of C. siamense strains. ** Significant at p < 0.01.

Figure 6

Conidial production and germination of C. graminicola strains. (A) Statistical analyses of the falcate conidium yield of C. graminicola strains. (B) Statistical analyses of the oval conidium yield of C. graminicola strains. ** Significant at p < 0.01. (C) Falcate conidium germination of C. graminicola strains. fc: falcate conidium, gt: germ tube, ap: appressorium.

Figure 7

Conidial production and germination of C. siamense strains. (A) Statistical analyses of the conidium yield of C. siamense strains. (B) Statistical analyses of the conidial germination rate. (C) Statistical analyses of the appressorium formation rate of C. siamense strains. ** Significant at p < 0.01. (D) Conidial germination of C. siamense strains. co: conidium, gt: germ tube, ap: appressorium.

Figure 8

Virulence assays. (A) Disease symptoms on maize leaves using conidial suspension or mycelial plug inoculation of CgrWT, the ΔCgrcon7 mutants, and the complementary strain. Statistical analyses of lesion diameters for conidial suspension (B) and mycelial plug (C) inoculation of the C. graminicola strains. (D) Disease symptoms on rubber tree leaves using conidial suspension or mycelial plug inoculation of CsWT, the ΔCscon7 mutants, and the complementary strain. 1: CK, 2: CsWT, 3: ΔCscon7-12, 4: ΔCscon7-15, 5: ΔCscon7-C. Statistical analyses of lesion diameters for conidial suspension (E) and mycelial plug (F) inoculation of the C. siamense strains. ** Significant at p < 0.01.

Figure 9

Functional classification and KEGG enrichment analyses of DEGs. (A) Functional classification of DEGs in the Cgrcon7-deletion mutant. (B) The top 20 enriched KEGG pathways of DEGs in the Cgrcon7-deletion mutant. (C) Functional classification of DEGs in the Cscon7-deletion mutant. (D) The top 20 enriched KEGG pathways of DEGs in the Cscon7-deletion mutant.