Functional characterization of salicylate hydroxylase from the fungal endophyte Epichloë festucae

Abstract

Epichloë spp. are symbiotic fungal endophytes of many cool season grasses. The presence of the fungal endophytes often confers insect, drought, and disease tolerance to the host grasses. The presence of the fungal endophytes within the host plants does not elicit host defense responses. The molecular basis for this phenomenon is not known. Epichloë festucae, the endophyte of Festuca rubra, expresses a salicylate hydroxylase similar to NahG from the bacterium Pseudomonas putida. Few fungal salicylate hydroxylase enzymes have been reported. The in planta expression of an endophyte salicylate hydroxylase raised the possibility that degradation of plant-produced salicylic acid is a factor in the mechanism of how the endophyte avoids eliciting host plant defenses. Here we report the characterization of the E. festucae salicylate hydroxylase, designated Efe-shyA. Although the fungal enzyme has the expected activity, based on salicylic acid levels in endophyte-free and endophyte-infected plants it is unlikely that expression of the endophyte salicylate hydroxylase is a factor in the lack of a host defense response to the presence of the fungal endophyte.

Figure 1. Reaction catalyzed by salicylate hydroxylase (E.C.1.14.12.1).

Figure 2. Comparison of deduced salicylate hydroxylase amino acid sequences from Epichloë festucae (accession KM400586), Ustilago maydis (accession XM_756284), and Pseudomonas putida (accession J05317).

The conserved functional domains are underlined. An asterisk indicates identical residues in all sequences, a “:” indicates strongly conserved residues (score > 0.5), and a “ . ” indicates weaker conserved residues (score < 0.5) (Thompson et al., 1997).

Figure 3. Gene structure of the E. festucae Rose City salicylate hydroxylase gene (accession KM400586), functional transcript, and alternatively spliced transcript variants.

The exons are indicated by boxes and the introns by lines. The positions of the premature termination sites in the alternatively spliced transcript variants are indicated by asterisks.

Figure 4. Rooted 50% majority rule maximum parsimony phylogenetic tree of Epichloë and related Clavicipitaceae spp. salicylate hydroxylase DNA coding sequences.

The Atkinsonella spp. sequences were designated as the outgroup for rooting the tree. The numbers at the nodes are the bootstrap percentages based on 1000 replications. The tree was based upon 1438 total characters, of which 717 were constant, 213 variable characters were parsimony uninformative, and 580 variable characters were parsimony informative. Available NCBI accession numbers of the contigs containing the sequences are: E. amarillans 200744, AFRF01001066.1; E. amarillans 4668, JFGZ01000088.1; E. baconii, JFGY01000338.1; E. festucae 2368, ADFL02000346.1; E. festucae Fl1, AFRX01000200.1; E. aotearoae, JFGX01000019.1; E. typhina E8, AMDI01000203.1; E. typhina subsp. poae, AFSE01001014.1; E. brachyelytri, AFRB01001961.1; E. elymi, AMDJ01000092.1; E. glyceriae, AFRG01000890.1; E. gansuensis, AFRE01000003.1 and AFRE01000959.1; E. gansuensis var. inebrians, AMDK01001066.1.

Figure 5. Expression of E. festucae Rose City salicylate hydroxylase in vivo.

PCR product of the E. festucae Rose City salicylate hydroxylase transcript using oligo(dT)-generated cDNA reverse-transcribed from total RNA of (1) E. festucae Rose City fungal endophyte grown in culture, and (2) endophyte-infected F. rubra leaf sheath tissue. Standard DNA markers are indicated by M.

Figure 6. Colorimetric plate assay confirming the enzymatic acitivity of the E. festucae Rose City salicylate hydroxylase.

The segmented LB plate was supplemented with 0.01 mM IPTG and 2.5 mM salicylic acid. (A) E. coli cells containing the pET21b(+) plasmid with the E. festucae Rose City salicylate hydroxylase. (B) E. coli cells containing the pET21b(+) vector only.

Figure 7. Expression of E. festucae Rose City salicylate hydroxylase in two E. coli expression strains.

SDS-PAGE analysis of E. festucae Rose City salicylate hydroxylase expressed in E. coli (A) BL21 CodonPlus RIPL (DE) cells showing Lane 1: insoluble pellet, Lane 2: 25μg total soluble protein, and Lane 3: proteins eluted from nickel-ion magnetic beads consisting mainly of the contaminating 44 kD bacterial protein, (B) ArcticExpress RP (DE) cells showing Lane 4: insoluble pellet, Lane 5: 25 μg total soluble protein, and Lane 6: 0.5 μg purified protein using TALON Metal Affinity Resin. Arrow indicates presence of the induced protein at the expected size of 48.3 kD. Asterisk is used to show the nickel-binding contaminating E. coli protein. The 60 kD bacterial chaperonin protein, Cpn60 expressed in E. coli ArcticExpress cells is indicated by ‡. Standard protein markers are indicated by M.

Figure 8. Salicylic acid levels in mature leaves and leaf sheaths of endophyte-free (S1139E-) and endophyte-infected (S1139Rose City and S1139Delaware) strong creeping red fescue plants.

Samples with different letters are significantly different at P < 0.05.