Magnaporthe oryzae Chloroplast Targeting Endo-β-1,4-Xylanase I MoXYL1A Regulates Conidiation, Appressorium Maturation and Virulence of the Rice Blast Fungus

Abstract

Endo-β-1,4-Xylanases are a group of extracellular enzymes that catalyze the hydrolysis of xylan, a principal constituent of the plant primary cell wall. The contribution of Endo-β-1,4-Xylanase I to both physiology and pathogenesis of the rice blast fungus M. oryzae is unknown. Here, we characterized the biological function of two endoxylanase I (MoXYL1A and MoXYL1B) genes in the development of M. oryzae using targeted gene deletion, biochemical analysis, and fluorescence microscopy. Phenotypic analysis of ∆Moxyl1A strains showed that MoXYL1A is required for the full virulence of M. oryzae but is dispensable for the vegetative growth of the rice blast fungus. MoXYL1B, in contrast, did not have a clear role in the infectious cycle but has a critical function in asexual reproduction of the fungus. The double deletion mutant was severely impaired in pathogenicity and virulence as well as asexual development. We found that MoXYL1A deletion compromised appressorium morphogenesis and function, leading to failure to penetrate host cells. Fluorescently tagged MoXYL1A and MoXYL1B displayed cytoplasmic localization in M. oryzae, while analysis of MoXYL1A-GFP and MoXYL1B-GFP in-planta revealed translocation and accumulation of these effector proteins into host cells. Meanwhile, sequence feature analysis showed that MoXYL1A possesses a transient chloroplast targeting signal peptide, and results from an Agrobacterium infiltration assay confirmed co-localization of MoXYL1A-GFP with ChCPN10C-RFP in the chloroplasts of host cells. MoXYL1B, accumulated to the cytoplasm of the host. Taken together, we conclude that MoXYL1A is a secreted effector protein that likely promotes the virulence of M. oryzae by interfering in the proper functioning of the host chloroplast, while the related xylanase MoXYL1B does not have a major role in virulence of M. oryzae.

Keywords: Chloroplast targeting peptide; Magnaporthe oryzae; Pathogenesis; Rice blast disease; Xylanases.

Fig. 1

Impact of MoXYL1 gene deletion on colony morphology and infectious growth of M. oryzae. a Strains of the indicated genotype were inoculated on CM media and photographed after 10-days of growth. b Statistical analysis of average colony diameter (mm) from three independent biological experiments with five replicates each. One-way ANOVA (non-parametric) was employed to assess statistical significance. Error bars account for standard deviation and asterisks represent the significant difference between wild type Guy11 and the mutant strain (p < 0.001). c Depicts drastically reduced ability of conidiophoregenesis of ΔMoxyl1A genes as compared to Guy11 at 12 h and 24 h interval. d Quantitation and statistical analysis of conidia production in ΔMoxyl1 strains relative to Guy11, obtained from cultures grown on Rice Bran Media, Straw Decoction and Corn media and CM-II media, respectively, from five independent biological experiments with five replicates. The data was analyzed with GraphPad Prism5; error bars represent the standard deviation, while a single asterisk (*) represent significant differences (p < 0.05) and double asterisks (**) represent significant differences (p < 0.001) according to ordinary one-way ANOVA

Fig. 2

Targeted gene replacement of MoXYL1A compromised turgor-mediated appressorium integrity and impaired the virulence of M. oryzae. a Showed hyphae-mediated virulence characteristics of the individual strains on intact and injured leaves of one-week-old, barley seedlings.induction of blast lesion was assessed at 7-dpi. Images are representative of three independent assays, each assay with three replicates. b Virulence bioassay conducted on 21-days-old rice seedlings using spore-drop inoculation method. Conidial suspensions were prepared as 1 × 10⁵ conidia mL⁻¹ in 0.2% Tween 20, for both the mutants and wild-type 20 µL. c Conidia-mediated pathogenicity/virulence characteristics ΔMoxyl1A, ΔMoxyl1B, and the wild-type on 21-days-old rice seedlings, through spray-inoculation with conidial suspensions (1 × 10⁵ conidia mL⁻¹ in 0.2% Tween 20). d The micrograph portrays the inability of ΔMoxyl1A and double deletion mutants to invade and colonize barley tissue at 48hpi. The absence of invasive hyphae were evident in barley cells inoculated with ΔMoxyl1A or the double deletion strain. In contrast, numerous invasive hyphae were seen in the leaves inoculated with either ΔMoxyl1B or Guy11. Images are representative of n = 2 independent biological replicates. Scale bar, 20 μm. e Appressoria were produced artificially on Thermo-fisher hydrophobic coverslips and observed at 8 hpi. Images show non-functional appressoria lacking melanin lining for the ΔMoxyl1A mutant. f Showed results from incipient cytorrhysis assays performed to evaluate the turgidity of appressorium form by conidia from the ΔMoxyl1A, ΔMoxyl1B, ΔMoxyl1A_Com., ΔMoxyl1B_Com., and the wild-type hydrophobic coverslips for 8-h appressorium formed were treated with 2 M glycerol solutions. Collapse appressorium were counted using the Olympus DP80 light microscope. Scale bar = 20 μm. g The bar graph showed results from the statistical evaluation of the proportion of collapsed appressorium recorded in the ΔMoxyl1A, ΔMoxyl1B, ΔMoxyl1A_Com., ΔMoxyl1B_Com., and the wild-type strains on hydrophobic coverslips for 8-h and treated independently with varying concentrations (1 M, 2 M, 3 M, and 4 M) of glycerol solutions during incipient cytorrhysis assays. Consistent results from three independent biological experiments with each consisting of five technical replicates were used for statistical analyses. For each independent biological experiment, 100 appressoria were counted (n = 100*3). treatments yielding significant difference (P ≤ 0.05) are denoted with asterisks “*”

Fig. 3

MoXYL1A and MoXYL1B mutants show varying degrees of sensitivity to cell wall perturbing agents. a Physical inhibitory effect of selected cell wall stress-inducing agents on the vegetative growth of the individual strains. The strain were cultured on CM media supplemented with 200 μg/mL Calcofluor White (CFW), 0.01% SDS or 200 μg/mL Congo Red (CR) for 10-days. b Quantification and statistical evaluation of the response of MoXYL1 single and double deletion mutants and the wild-type strain to different cell wall stress inducing reagents. The inhibition data was generated from five independent biological experiments with five technical replicates each. One-way ANOVA (non-parametric) statistical analysis was carried out with GraphPad Prism8 and Microsoft Excel. Error bars represent standard deviation. Inhibition rate was calculated as a percentage = (the diameter of control − the diameter of treatment)/ (the diameter of control) × 100. Single asterisk represents a significant difference (p < 0.05)

Fig. 4

Subcellular localization of the relative expression xylanases at different stages of M. oryzae -host interaction. a Localization of MoXYL1A in the aberant conidia, conidia germination and appressorium formation stages of M. oryzae was determined by transforming a MoXYL1A-GFP fusion construct into the protoplast of the wild type strain Guy11 and examining fungal cells using the Scale bar = 20 µm. DIC indicates bright field illumination. GFP was excited at 488 nm. b Localization of MoXYL1B was assessed as in (a). MoXYL1B-GFP signal is evident in the conidium and appressorium. Scale bar = 20 µm. c In-planta expression of MoXYL1A and MoXYL1B transcripts during distinct stages of host–pathogen interaction was assessed by qRT-PCR. Vegetative hyphae were used as a control stage and the expression level of MoXYL1A and MoXYL1B at the hyphal stage was set to 1. Error bars represent standard deviation (SD). SD was calculated from three independent biological replicates along with three technical replicates. (*, P < 0.05 by t-test)

Fig. 5

MoXYL1A accumulated at the Chloroplasts of barley and tobacco seedlings. a Showed the localization pattern of MoXYL1A-GFP during M. oryzae interaction with barley host during early (24–48-hpi), and late (72–96-hpi) stages of pathogen-host interaction. b The micrograph revealed the accumulation of MoXYL1A-GFP to the chloroplast of leaf epidermal tissues of barley leaves at 72-hpi. Scale bar = 10 µm. c and d The micrograph confirmed the co-expression of (MoXYL1A-GFP) and the chloroplast marker (ChCpn10) in the chloroplas of agro-infiltrated tobacco seedlings. Scale bar = 10 µm. e Showed distortions in the localization pattern of MoXYL1A-^∆ctp-GFP. The MoXYL1A-^∆ctp-GFP signals accumulated at the membrane or extracellular regions of agrobacterium infiltrated tobacco seedlings at 48-hpi. GFP was excited at 488 nm and RFP was excited at 561 nm. Scale bar = 20 µm

Fig. 6

Comparative sequence features of M. oryzae XYL1s and expression dynamics for pathogenicity-related genes in rice seedlings inoculated with strains lacking MoXYL1s. a and b Comparative alignment results for MoXYL1A and MoXYL1B and detail sequence architecture for MoXYL1A and MoXYL1B. Sequences shaded in Blue denote the secretion signal peptides (SP), sequences shaded in Red denote the chloroplast targeting peptide (cTP), sequences shaded in Green denote the conserved Glyco-hydro_11 domain motif, and the cleavage site is denoted as (CS) c Showed the relative expression (in folds) of genes coding for pathogenicity-related proteins in rice blast susceptible CO39 cultivar inoculated with ΔMoxyl1A, ΔMoxyl1B, ΔMoxyl1A_Com., ΔMoxyl1B_Com., and the wild-type at 12-hpi. Error bars represent standard deviation (SD). SD was calculated from three independent biological replicates along with three technical replicates. (*, P < 0.05 by t-test)