RNA interference of endochitinases in the sugarcane endophyte Trichoderma virens 223 reduces its fitness as a biocontrol agent of pineapple disease

publication-status: PublishedThe sugarcane root endophyte Trichoderma virens 223 holds enormous potential as a sustainable alternative to chemical pesticides in the control of sugarcane diseases. Its efficacy as a biocontrol agent is thought to be associated with its production of chitinase enzymes, including N-acetyl-β-D-glucosaminidases, chitobiosidases and endochitinases. We used targeted gene deletion and RNA-dependent gene silencing strategies to disrupt N-acetyl-β-D-glucosaminidase and endochitinase activities of the fungus, and to determine their roles in the biocontrol of soil-borne plant pathogens. The loss of N-acetyl-β-D-glucosaminidase activities was dispensable for biocontrol of the plurivorous damping-off pathogens Rhizoctonia solani and Sclerotinia sclerotiorum, and of the sugarcane pathogen Ceratocystis paradoxa, the causal agent of pineapple disease. Similarly, suppression of endochitinase activities had no effect on R. solani and S. sclerotiorum disease control, but had a pronounced effect on the ability of T. virens 223 to control pineapple disease. Our work demonstrates a critical requirement for T. virens 223 endochitinase activity in the biocontrol of C. paradoxa sugarcane disease, but not for general antagonism of other soil pathogens. This may reflect its lifestyle as a sugarcane root endophyte

Towards a Comprehensive Search of Putative Chitinases Sequences in Environmental Metagenomic Databases

Chitinases catalyze the hydrolysis of chitin, a linear homopolymer of β-(1,4)-linked N-acetylglucosamine. The broad range of applications of chitinolytic enzymes makes their identification and study very promising. Metagenomic approaches offer access to functional genes in uncultured representatives of the microbiota and hold great potential in the discovery of novel enzymes, but tools to extensively explore these data are still scarce. In this study, we develop a chitinase mining pipeline to facilitate the comprehensive search of these enzymes in environmental metagenomic databases and also to explore phylogenetic relationships among the retrieved sequences. In order to perform the analyses, UniprotKB fungal and bacterial chitinases sequences belonging to the glycoside hydrolases (GH) family-18, 19 and 20 were used to generate 15 reference datasets, which were then used to generate high quality seed alignments with the MAFFT program. Profile Hidden Markov Models (pHMMs) were built from each seed alignment using the hmmbuild program of HMMER v3.0 package. The best-hit sequences returned by hmmsearch against two environmental metagenomic databases (Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis—CAMERA and Integrated Microbial Genomes—IMG/M) were retrieved and further analyzed. The NJ trees generated for each chitinase dataset showed some variability in the catalytic domain region of the metagenomic sequences and revealed common sequence patterns among all the trees. The scanning of the retrieved metagenomic sequences for chitinase conserved domains/signatures using both the InterPro and the RPS-BLAST tools confirmed the efficacy and sensitivity of our pHMM-based approach in detecting putative chitinases sequences. These analyses provide insight into the potential reservoir of novel molecules in metagenomic databases while supporting the chitinase mining pipeline developed in this work. By using our chitinase mining pipeline, a larger number of previously unannotated metagenomic chitinase sequences can be classified, enabling further studies on these enzymes

Targeted gene deletion of the <i>N-</i>acetyl-ß-D-glucosaminidase-encoding genes <i>nag1</i> and <i>nag2</i> by using the split-marker strategy.

<p>Schematic representations of vector construction are shown in (a) for the single knockout strains, and (b) for the double knockout strains (a) Primers 1F/2R and 3F/4R amplify 1kb target gene flanking sequences. Primers 2R and 3F are hybridised once the 5′ ends complement the M13F and M13R sequences, respectively. For <i>nag1</i>, the primer pairs Chito2_50.1/Chito2_M13F and Chito2_M13R/Chito2_30.1 were used and, for <i>nag2,</i> the primer pairs HEX1_Fw/HEX1.M13F_Rev and HEX2.M13R_Fw/HEX2_Rev were used. Primer pairs M13F/HYsplit and M13R/YGsplit amplify the ‘HP’ and ‘PH’ marker fragments, respectively. Two separate PCR reactions (1F/HYsplit) and (YGsplit/4R) fuse the flanking sequences to the 5′ ‘HP’ or 3′ ‘PH’ fragments of the hygromycin resistance gene <i>hph</i>. Similar steps were used for double knockout mutant generation (b), using a <i>nag2</i> deletion mutant for transformation with a second selectable marker <i>ilv1</i> bestowing resistance to sulfonylurea. In this case, the primer pairs used in the first round PCR reactions were Chito2_M13R/Chito2_30.1 and Chito2_50.1/Chito2_M13F for amplification of the 1 kb <i>nag1</i> flanking sequences and ILsplit/M13R and M13F/LVsplit for amplification of the ‘IL’ and ‘LV1’ fragments, respectively. Second round PCR reactions were performed with the primer pairs Chito2_30.1/ILsplit and LVsplit/Chito2_50.1. Primer sequences are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047888#pone-0047888-t001" target="_blank">Table 1</a>. (c), Putative ?<i>nag2</i> transformants. The lane indicated by Wt consists of wild type <i>Tv.</i>223 DNA and lanes 1–14 contain DNA of putative ?<i>nag2</i> knockouts. The ?<i>Tvnag2</i> mutant in lane 10 (confirmed by the absence of a band) was selected for <i>nag2</i> loss-of-function studies and for the generation of ?<i>Tvnag1</i>?<i>Tvnag2</i> mutants. (d) Putative ?<i>nag1</i> transformants. The lane indicated by Wt consists of wild type <i>Tv.</i>223 DNA and lanes 1–13 contain DNA of putative ?<i>nag1</i> knockouts. The ?<i>Tvnag1</i> mutant in lane 5 (confirmed by the absence of a band) was selected for <i>nag1</i> loss-of-function studies. (e) Putative ?<i>Tvnag1</i>?<i>Tvnag2</i> transformants. Lane 1 contains DNA of the ?<i>Tvnag2</i> mutant (lane 10 in (c)) used for the generation of the double mutants. Lane 2 contains DNA of the ?<i>Tvnag1</i> mutant from lane 5 in (d). Lanes 3–12 contain DNA of putative double mutants. The double mutant, ?<i>Tvnag1</i>?<i>Tvnag2</i>, in lane 3 was selected for loss-of-function studies.</p

Detection of chitinolytic activities in culture filtrates of the <i>T. virens</i> strains.

<p><i>N-</i>acetyl-ß-D-glucosaminidase, chitobiosidase and endochitinase activities of the wild type strain <i>Tv.</i>223 and the five chitinase-deficient mutants ?<i>Tvnag1</i>, ?<i>Tvnag2</i>, ?<i>Tvnag1</i>?<i>Tvnag2,</i> S.pNO8::<i>hph,</i> and S.pOf28::<i>hph</i> were determined by enzymatic hydrolysis of the chitinase-specific substrates 4-Nitrophenyl <i>N</i>-acetyl-ß-D-glucosaminide, 4-Nitrophenyl N,N’-diacetyl-ß-D-chitobioside and 4-Nitrophenyl ß-D-N,N’,N’’-triacetylchitotriose, respectively. Substrate hydrolysis releases <i>p</i>-nitrophenol which, upon ionization in basic pH, can be measured colorimetrically at 405 nm. Protein concentrations of the extracts were adjusted to 20 µg.mL⁻¹ prior to enzyme assays. Histograms are the means of three replicate values with standard deviations. One unit of the activity releases 1.0 µmole of <i>p-</i>nitrophenol from the appropriate substrate per minute at pH 4.8 and 37°C. Letters denote the results of a <i>t</i>-test for comparison of means. Bars with different letter(s) are significantly different at 95% confidence level.</p

Colony diameters and conidiation of wild type <i>Tv.</i>223 and the chitinase-deficient mutants.

*<p>Each figure is the mean diameter of three replicate cultures.</p><p>Based on ANOVA (<i>P</i><0.05), there were no significant differences in hyphal growth and conidiation between strains.</p

RNA-mediated silencing vectors and Southern blot analysis.

<p>(<b>a</b>) Schematic representation of the vector pSilent1-endochi, derived from pSilent-1 (Nakayashiki <i>et al.</i>, 2005) by two asymmetric clonings of a 491 bp <i>Tvech2</i> fragment. (<b>b</b>) Schematic representation of the vector pSilent1-ToxA, derived from pSilent-1 (Nakayashiki <i>et al.</i>, 2005) by replacement of the trpC promoter by ToxA followed by two asymmetric clonings of a 491 bp <i>Tvech2</i> fragment. Amp^r, ampicillin-resistant gene; Hyg^r, hygromycin-resistant gene; endochi, 491 bp <i>Tvech2</i> fragment; IT, intron 2 of the cutinase (CUT) gene from <i>Magnaporthe oryzae</i>; PtrpC, <i>Aspergillus nidulans</i> trpC promoter; and TtrpC, <i>A. nidulans</i> trpC terminator. Southern blots are show below the vector maps. Genomic DNA was digested with <i>Sac</i>I + <i>Kpn</i>I and probed with a 1 kb fragment amplified from the hygromycin cassette with the primers Hyg probe 2F/Hyg probe 3R. In (<b>a</b>): lane 1 contains DNA of the wild type strain <i>Tv.</i>223 and lanes 2–11 contain DNA of the putative transformants S.pNO6::<i>hph</i>, S.pNO8::<i>hph</i> (used in the loss-of-function studies), S.pNO15::<i>hph</i>, S.pNO23::<i>hph</i>, S.pNO28::<i>hph</i>, S.pNO3::<i>hph</i>, S.pNO5::<i>hph</i>, S.pNO9::<i>hph</i>, S.pNO13::<i>hph</i> and S.pNO31, respectively. In (<b>b</b>): lane 1 contains DNA of the transformant S.pOf28::<i>hph</i> (used in the loss-of-function studies), lane 2 contains the DNA of the wild type strain <i>Tv.</i>223 and lanes 3–12 contain DNA of the putative transformants S.pOf31::<i>hph</i>, S.pOf32::<i>hph</i>, S.pOf39::<i>hph</i>, S.pOf11::<i>hph</i>, S.pOf21::<i>hph</i>, S.pOf17::<i>hph</i>, S.pOf36::<i>hph</i>, S.pOf40::<i>hph</i>, S.pOf15::<i>hph</i> and S.pOf8::<i>hph</i>, respectively. The presence of a band confirms insertion of the vector.</p

Northern blot analysis.

<p>The effects of insertion of the silencing vectors pSilent1-endochi and pSilent1-ToxA on the expression of three chitinase genes (<i>Tvech1, Tvech2</i> and <i>Tvech3</i>) in <i>T. virens</i> transformants are shown in (<b>a</b>) detection of <i>Tvech1</i> transcripts, (<b>b</b>) detection of <i>Tvech2</i> transcripts and, (<b>c</b>) detection of <i>Tvech3</i> transcripts. Transformants containing pSilent1-endochi vector have the prefix S.pNO and the ones containing pSilent1-ToxA have the prefix S.pOf. Twenty micrograms of total RNA were separated on formaldehyde-agarose gels, transferred to nylon membranes and probed with a 1 kb gene-specific fragment. Equal loading of total RNA was estimated by ethidium bromide staining of rRNA.</p

Biological control of soil-borne pathogens.

<p>(<b>a</b>) Biocontrol of pineapple disease of sugarcane caused by the soil-borne pathogen <i>Ceratocystis paradoxa.</i> Germination of sugarcane setts was determined in compost infested with the pathogen <i>C. paradoxa</i> and in mixed-species microcosms containing the pathogen and chitinase-deficient mutants. Percentage germination was measured weekly, over a 6 week period. The control consisted of sugarcane setts planted in uninfested compost. Each point is the mean of twelve replicates and percentages were converted to arc sin⁻¹ values for statistical analysis by <i>t</i>-test. Points with different letters are significantly different at 95% confidence level, considering each week separately. (<b>b</b>) Biocontrol of pre-emergence damping-off disease of lettuce caused by the pathogen <i>Sclerotinia sclerotiorum</i>, Germination of lettuce seeds was determined in peat-based microcosms infested with the pathogen <i>S. sclerotiorum</i> and in mixed-species microcosms containing the pathogen and chitinase-deficient mutants. Percentage germination was measured 10 days after sowing. The control consisted of lettuce seeds planted in uninfested peat. Each point is the mean of three replicates (each consisting of 25 lettuce seed) and percentages were converted to arc sin⁻¹ values for statistical analysis by Tukey test for comparison of means. Histograms with different letters are significantly different at 99% confidence level. (<b>c</b>) Biocontrol of bean rot caused by the pathogen <i>Rhizoctonia solani</i>. Germination of bean seeds was determined in compost infested with the pathogen <i>R. solani</i> and in mixed-species microcosms containing the pathogen and chitinase-deficient mutants. Percentage germination was measured 20 days after sowing. The control consisted of bean seeds planted in uninfested compost. Each point is the mean of six replicates and percentages were converted to arc sin⁻¹ values for statistical analysis by Tukey test for comparison of means. Histograms with different letters are significantly different at 95% confidence level.</p

Details of PCR primers used in this study.

<p>LF – left flank.</p><p>RF – right flank.</p