A Botrytis cinerea KLP-7 Kinesin acts as a Virulence Determinant during Plant Infection

Abstract

Botrytis cinerea is a necrotrophic pathogen that infects many important crops. In an attempt to unravel some novel factors that govern pathogenicity in B. cinerea, Agrobacterium tumefaciens mediated transformation (ATMT) was deployed, and a number of tagged transformants were generated. Among these, a mutant, BCM-29 exhibited slower growth rate, reduced conidia size, conidiation and penetration. The mutant was also defective in secretion of oxalic acid (OA) and exhibited reduced activities of polygalacturonase (PG) and pectin methyl esterases (PME). TAIL-PCR followed by BLAST search identified the tagged gene as KLP-7 that encodes for kinesin. Targeted deletion of KLP-7 resulted in several folds decrease in virulence of mutants as compared to WT, while complementation of the gene helped in rescue of virulence traits. This is the first time when a unique kinesin KLP-7 that is mainly found in the phylum Pezizomycotina has been linked to virulence in B. cinerea.

Figure 1

Optimization of co-cultivation conditions for Agrobacterium tumefaciens mediated transformation of B. cinerea. (A) Concentration of acetosyringone (µM); (B) Temperature of co-cultivation; (C) pH of Induction medium; (D) Filter discs.

Figure 2

An abnormal T-DNA integration in KLP-7. (A) Hygromycin phosphotransferase (hph) gene insertion in BCM-29. The expected PCR product size is 1.1 kb. Lane 1, Ladder of 10 kb; lane 2, WT, lane 3, BCM-29, lane 4, positive control (PC) (hph) (B). Southern hybridization showing the integration of 4.1 kb fragment of hph gene in genome of BCM 29. Total genomic DNA was digested with XhoI and probed with the hph probe; (C) Schematic presentation integration of T-DNA in BCM 29. Specific primers were used for the confirmation of T-DNA insertion shown by arrows. The T-DNA insertion point is 116 bp from KLP-7 start codon (D) Insertion of T-DNA in B. cinerea genome. Junction site are shown in bold letters.

Figure 3

Alignment of the deduced amino acid sequence of KLP-7. B. cinerea AAO59283 (BC) with Sclerotinia sclerotiorum XP_001598213 (SS); Fusarium verticillioides EWG48122 (FV), Magnaporthe oryzae XP_003715125 (MO), Neurospora crassa XP_961491(NC), Aspergillus niger XP_001401241(AN), Bipolaris maydis AAO59294 (BM) by using MULTALIN. The high degree of sequence conservation at each position of amino acids is shown in blue, and low consensus amino acids are shown in red. Signature tag sequences of conserved motor domain of KIF1A/UNC4 are shown in box.

Figure 4

Phylogenetic analysis: The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 8.63069435 is shown. Percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. Analysis involved 55 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 78 positions in the final dataset. Evolutionary analyses were conducted in MEGA7. Member of different groups were marked with different shape i.e. Δ: fungi, □: animals, ◊: Plants, ○: Insects, ∇: Bacteria and B. cinerea kinesin protein is marked with filled triangle (▲) shape to display its position.

Figure 5

Identification of a KLP-7 deletion mutant in B. cinerea and relative gene expression analysis. (A) The KLP-7 deletion cassette (3,571 bp) with the hph cassette replaced the bcKLP-7 ORF by double crossing over. Flanking genomic regions are shown by white box (B) Gel electrophoretic profile showing amplification of KLP-7 gene using gene specific primer pair in WT and mutants. Lane1: WT, Lane 2: complemented mutant bcKLP-7 ^COM, Lane 3: T-DNA tagged mutant BCM-29, Lane 4: deletion mutant ∆bcKLP-7 (C) Fluorescence microscopy for the expression analysis of GFP in (i) WT, (ii) BCM-29 and (iii) bcKLP-7 ^COM (D) Western blot analysis for GFP expression in WT, BCM-29 and bcKLP-7 ^COM (E) Expression analysis of KLP-7 in WT, BCM-29, bcKLP-7 ^COM and in ∆bcKLP-7. Relative expression levels were normalized to the mean of the expression of reference gene (actin). The expression of WT was set to 1. Data is presented as mean ± SD (n = 3). Bars showing different letters indicate significant differences between WT and mutants according to the Duncan’s multiple comparison tests (P < 0.05).

Figure 6

Radial growth, sclerotia formation, mycelia dry weight assay and scanning electron microscopy of conidia and hyphal structure. (A) Colony morphology and development of sclerotia. The wild type strain, BCM-29, bcKLP-7 ^COM and ∆bcKLP-7 were incubated on PDA medium at 22 °C for 3 weeks in darkness. Arrow indicates the melanised structure, sclerotia; (B) Colony diameters of WT, BCM-29, bcKLP-7 ^COM and ∆bcKLP-7 after 7 days growth on PDA medium; (C) The dry weight mycelium assay for WT and mutants. Cultures were incubated in Potato dextrose broth for seven days. Data is presented as mean ± SD. Bars showing different letters indicate significant differences between WT and mutants according to the Duncan’s multiple comparison tests (P < 0.05); (D) Conidia morphology and size observed under scanning electron microscope; (E) Hyphal structure of indicated strains observed under a scanning electron microscope. Scale bar represents 10 µm. Data is presented as mean ± SD. Bars showing different letters indicate significant differences between WT and mutants according to the Duncan’s multiple comparison tests (P < 0.05).

Figure 7

Infection related morphogenesis and role of virulence components. (A) Virulence assay on detached tomato leaves. Spore suspension (10⁵ spores/mL) was drop inoculated on upper surface of sterilized leaves of tomato under in vitro conditions; (B) Lesion diameter of WT and corresponding mutants observed on tomato leaves. Observations were made four days post inoculation; (C) Disease severity on chickpea plants to screen impaired virulence in B. cinerea mutants (T-DNA tagged mutant, complemented mutant and gene deletion mutant) in comparison to WT. (D) Conidia germination rate of respective strains on onion epidermis after incubation at 22 °C was evaluated after 24 hpi, (E) Penetration ability of strains was assessed at 48 hpi. Data are presented as mean ± SD. Bars showing different letters indicate significant differences according to the Duncan’s multiple comparison tests (P < 0.05). (F) Tissue colonization of onion epidermis observed after 48 h of inoculation. Spore suspension (10⁵ spores/mL) was drop inoculated on hydrophobic surface of sterilized tissue. Epidermis was stained with cotton blue in lactophenol for microscopic examination. Scale bars represent 20 µm. Arrows indicate penetration of hyphae in epidermis. Experiment was performed in five replicates and was repeated three times.

Figure 8

Virulence factors determination. (A) Oxalic acid concentration in culture filtrates of WT, BCM-29, bcKLP-7 ^COM and ∆bcKLP-7 incubated for 10 days; (B) and (C). The activities of cell wall degrading enzymes PG and PME in WT and corresponding mutants incubated for ten days. Data is presented as mean ± SD. Bars showing different letters indicate significant differences according to the Duncan’s multiple comparison tests (P < 0.05).