Overexpression of a novel biotrophy-specific Colletotrichum truncatum effector, CtNUDIX, in hemibiotrophic fungal phytopathogens causes incompatibility with their host plants

Abstract

The hemibiotrophic fungus Colletotrichum truncatum causes anthracnose disease on lentils and a few other grain legumes. It shows initial symptomless intracellular growth, where colonized host cells remain viable (biotrophy), and then switches to necrotrophic growth, killing the colonized host plant tissues. Here, we report a novel effector gene, CtNUDIX, from C. truncatum that is exclusively expressed during the late biotrophic phase (before the switch to necrotrophy) and elicits a hypersensitive response (HR)-like cell death in tobacco leaves transiently expressing the effector. CtNUDIX homologs, which contain a signal peptide and a Nudix hydrolase domain, may be unique to hemibiotrophic fungal and fungus-like plant pathogens. CtNUDIX lacking a signal peptide or a Nudix motif failed to induce cell death in tobacco. Expression of CtNUDIX:eGFP in tobacco suggested that the fusion protein might act on the host cell plasma membrane. Overexpression of CtNUDIX in C. truncatum and the rice blast pathogen, Magnaporthe oryzae, resulted in incompatibility with the hosts lentil and barley, respectively, by causing an HR-like response in infected host cells associated with the biotrophic invasive hyphae. These results suggest that C. truncatum and possibly M. oryzae elicit cell death to signal the transition from biotrophy to necrotrophy.

Fig 1

Bootstrapped neighbor-joining tree of NUDIX effectors. (A) Domain and motif organization of CtNUDIX. (B) Effectors containing a signal peptide at the N terminus and a Nudix hydrolase domain in the C-terminal region were identified by searching the genomes of hemibiotrophic fungal and oomycete phytopathogens. The phylogenetic tree was constructed with peptide sequences using MEGA 4.1 software. NUDIX effectors prefixed with Ct, Cg, Ch, Mo, and Pi correspond to C. truncatum, C. graminicola, C. higginsianum, M. oryzae, and P. infestans, respectively. The asterisk represents a gene duplication event in M. oryzae. SP-HMM (signal peptide probability based on the hidden Markov model) was predicted using the SignalP 3.0 server. The best BLASTP match (E value ≤ 10¹⁵) for each protein is listed in the annotation column. Putative NUDIX motifs were isolated by the NCBI conserved domain search server (see Fig. S1 in the supplemental material).

Fig 2

CtNUDIX transcripts are exclusively accumulated in the in planta late biotrophic phase of the infection. (A) Trypan blue-stained in planta fungal structures. Lentil leaflets were droplet inoculated with C. truncatum isolate CT-21 and harvested at 16, 44, and 68 hpi, which represent appressorium penetration and the in planta biotrophic and necrotrophic phases of C. truncatum, respectively. M, mycelia; C, ungerminated conidia; M+CW, cell wall-treated mycelia; A, appressorium; PP, penetration peg; PH, primary hyphae; SH, secondary hyphae. Scale bars, 10 μm. (B) Northern blot analysis of C. truncatum gene expression (CtNUDIX and 60S ribosomal protein-encoding genes) in the infection time course and in vitro-grown cell types. Total RNA samples (20 μg/lane) were fractioned on a formaldehyde-agarose denaturing gel and transferred to a nylon membrane. The blots were then probed with a 593-bp 60S ribosomal gene fragment (as a marker to evaluate the in planta biomass of C. truncatum) and a 672-bp CtNUDIX cDNA fragment. An ethidium bromide-stained agarose gel prior to transfer to a nylon membrane is shown as a loading control. (C) Quantitative RT-PCR analysis of CtNUDIX transcript levels in fungal cell types of C. truncatum, such as mycelia and conidia, and an infection time course. The transcript levels of CtNUDIX were normalized to a housekeeping gene, the 60S ribosomal gene. All relative expression values of genes are reported as means ± standard errors (calculated from 3 biological replications and 3 technical replications/biological replication) on a log₂ scale.

Fig 3

Transient expression of CtNUDIX in tobacco. Shown are symptoms observed on tobacco leaves following the infiltration of recombinant A. tumefaciens strains carrying the binary vector pGR106 (negative control), pGR106-CtNUDIX, pGR106-CtNUDIXΔSP, pGR106-CtNUDIXΔNM, and pGR106-INF1 (positive control) at 5 dai. The assay was replicated independently 10 times. The presence (+) or absence (−) of macroscopic cell death observed in the infiltration zones is shown.

Fig 4

Localization of CtNUDIX:eGFP fusion protein in N. tabacum. (A) CtNUDIX:eGFP fusion construct pGR106-CtNUDIX:eGFP. The expression of the CtNUDIX:eGFP fusion construct was under the control of the cauliflower mosaic virus 35S promoter (35S::CtNUDIX:eGFP). (B) Leaf cells transiently expressing CtNUDIX:eGFP fusion proteins were visualized under a confocal microscope using an argon laser. Scale bar, 50 μm. DIC, differential interference contrast. (C and D) PI-labeled (C) and FM4-64-labeled (D) plasmolyzed N. tabacum leaf tissues expressing a 35S::CtNUDIX:eGFP construct were examined under a confocal microscope for GFP (green) and PI (red) or FM4-64 (red) fluorescence detection. The arrows and arrowheads indicate the cell wall and plasma membrane, respectively. The areas outlined by dashed lines represent dead cells. Scale bar, 25 μm.

Fig 5

Overexpression of CtNUDIX in C. truncatum causes incompatibility with its host, lentil. (A) An overexpression construct, RP27::CtNUDIX. The expression of CtNUDIX was under the control of the strong constitutive expression promoter RP27 and the TrpC terminator. (B) qRT-PCR analysis of CtNUDIX overexpression transformants of C. truncatum. (C) Three-week-old susceptible L. culinaris cv. Eston plants were sprayed with conidial suspensions (4 × 10⁴ conidia ml⁻¹) of CT-21, CtNUDIX/Ct-7, and CtNUDIX/Ct-10 and photographed at 7 dpi. (D) Microscopic evaluation of lentil leaf cells infected by CT-21 and overexpression mutants at 7 dpi. Ac, acervulus; PH, primary hyphae; SH, necrotrophic hyphae. The asterisks represent appressoria. Scale bars, 25 μm.

Fig 6

Heterologous expression of CtNUDIX in M. oryzae causes incompatibility with its host, barley. (A) Northern blot analysis of CtNUDIX-expressing M. oryzae transformants. RNA gel blots prepared from total mycelial RNA of transformants were hybridized with a 672-bp CtNUDIX cDNA fragment. Ethidium bromide staining of total RNA is shown as a loading control. (B) Ten-day-old leaves of susceptible H. vulgare cv. CDC Silky were droplet inoculated with P131, CtNUDIX/Mo-9, and CtNUDIX/Mo-17. Fifteen microliters of conidial suspension (4 × 10⁴ conidia ml⁻¹) were spotted onto the adaxial sides of leaves without damaging the surface and photographed at 10 dpi. The arrowheads mark typical necrotic-blast lesions on leaves inoculated with P131, whereas there is discoloration (light brown) on leaves inoculated with CtNUDIX overexpression strains. (C) Microscopic evaluation of barley cells infected by P131 and overexpression mutants CtNUDIX/Mo-9 and CtNUDIX/Mo-17 (10 dpi). Scale bars, 10 μm. A, appressorium; BIH, biotrophic invasive hyphae; NIH, necrotrophic invasive hyphae.