Divergent LysM effectors contribute to the virulence of <i>Beauveria bassiana</i> by evasion of insect immune defenses

<div><p>The lysin motif (LysM) containing proteins can bind chitin and are ubiquitous in various organisms including fungi. In plant pathogenic fungi, a few LysM proteins have been characterized as effectors to suppress chitin-induced immunity in plant hosts and therefore contribute to fungal virulence. The effector mechanism is still questioned in fungus-animal interactions. In this study, we found that LysM proteins are also present in animal pathogenic fungi and have evolved divergently. The genome of the insect pathogen <i>Beauveria bassiana</i> encodes 12 LysM proteins, and the genes were differentially transcribed by the fungus when grown in different conditions. Deletion of six genes that were expressed by the fungus growing in insects revealed that two, <i>Blys2</i> and <i>Blys5</i>, were required for full fungal virulence. Both proteins could bind chitin and Blys5 (containing two LysM domains) could additionally bind chitosan and cellulose. Truncation analysis of Blys2 (containing five LysM domains) indicated that the combination of LysM domains could determine protein-binding affinity and specificity for different carbohydrates. Relative to the wild-type strain, loss of <i>Blys2</i> or <i>Blys5</i> could impair fungal propagation in insect hemocoels and lead to the upregulation of antifungal gene in insects. Interestingly, the virulence defects of Δ<i>Blys2</i> and Δ<i>Blys5</i> could be fully restored by complementation with the Slp1 effector from the rice blast fungus <i>Magnaporthe oryzae</i>. In contrast to Slp1 and Blys2, Blys5 could potentially protect fungal hyphae against chitinase hydrolysis. The results of this study not only advance the understanding of LysM protein evolution but also establish the effector mechanism of fungus-animal interactions.</p></div

Protein localization assays.

<p><b>A</b>. Localization of Blys2 on the fungal cell walls of different type cells. Full length <i>Blys2</i> was fused in frame with a <i>GFP</i> gene and the cassette was controlled by the <i>GpdA</i> gene promoter for transformation of the WT strain of <i>B</i>. <i>bassiana</i>. <b>B</b>. Cytosolic localization of Blys2 without signal peptide (Blys2-SP) in different cells. The truncated <i>Blys2</i> without signal peptide sequence was fused in frame with a <i>GFP</i> gene and the cassette was controlled by the <i>GpdA</i> gene promoter for transformation of the WT strain of <i>B</i>. <i>bassiana</i>. <b>C</b>. Cytosolic localization of GFP protein. The <i>GFP</i> gene was controlled by the <i>GpdA</i> gene promoter for transformation of the WT strain of <i>B</i>. <i>bassiana</i>. <b>D</b>. Localization of Slp1 on the cells walls of <i>B</i>. <i>bassiana</i> cells. Full length <i>Slp1</i> of <i>M</i>. <i>oryzae</i> was fused in frame with a <i>GFP</i> gene and the cassette was controlled by the <i>GpdA</i> gene promoter for transformation of the WT strain of <i>B</i>. <i>bassiana</i>. The cells of different mutants were examined and photographed under the 100 × field of a confocal microscope. CO, conidia; HY, hyphae harvested from the PDA plate for three days; BL, blastospore; MY, mycelia harvested from SDB for three days; HB, hyphal body harvested from insect hemocoels. CW, Calcofluor White. Bar, 2 μm.</p

Flowchart of the experimental procedure of the screen.

<p>A <i>X. tropicalis</i> library of unique, full-length clones has been established based on sequence comparison and clustering of over 1,220,000 ESTs, and rearrayed in a 96-well plate format. Pools of 8 mRNAs were prepared from pooled bacteria culture and <i>in vitro</i> transcription. Then <i>in vitro</i> transcribed mRNA pools were injected into fertilized <i>X. laevis</i> embryos at 1–2 cell stage. After microinjection, injected embryos were collected at stage 8 (blastula), stage 10.5 (gastrula), and stage 14 (neurula). Protein extracts from embryos were loaded onto SDS-PAGE for subsequent Western blot analysis. Antibodies used include anti-phospho-Smad1/5/8, anti-phospho-Smad2, anti-phospho-Akt, and anti-phospho-Erk. Once a potential active pool was identified, the pool was de-convoluted and single molecule injection was performed to identify the active molecule.</p

Selection of the clones used to validate the screen strategy.

<p>Selection of the clones used to validate the screen strategy.</p

Whole-mount <i>in situ</i> hybridisation images on clones with regionalised expression patterns.

<p>For each clone the corresponding clone number and <i>Xenopus</i> gene symbol are shown. Vegetal view (stage 10.5 except for <i>foxh1</i>, which is side view); dorsal view (stage 15 and 20, posterior is up); lateral view (stage 30, anterior is to the left).</p

Kinetics of the activation of signalling molecules during early <i>Xenopus</i> development.

<p><i>X. laevis</i> embryos were collected at the time indicated and subjected to Western blot analysis. Membranes were probed with anti-phospho-Smad1/5/8 (pSmad1) antibody for monitoring BMP activity, anti-phospho-Smad2 (pSmad2) antibody for TGF-β/Nodal signalling, anti-phospho-Erk (pErk) for MAPK/Erk signalling and anti-phospho-Akt (pAkt) for PI3K/Akt signalling. Anti- Smad2, anti- Akt, and anti-Erk were used as loading controls to ensure all lanes have been loaded equally.</p

Proof of principle of the screen.

<p>(A) 12 pools, each one with one clone of known activity, were selected from the full-length EST library and injected into embryos as described. Protein extracts from collected embryos were subjected to Western blot using indicated antibodies to observe phosphorylation changes of specific signalling molecules at blastula and gastrula stages. Note the reduction of phospho-Smad2 activity at gastrula stage on pool 2, and reduction of phospho-Smad1/5/8 activity on pool 11. (B) De-convolution of pool 2. <i>cerberus</i> is identified as a negative regulator of Smad2 (pSmad2, lower panel) but not of Smad1 (pSmad1, upper panel) phosphorylation at gastrula stage. (C) De-convolution of pool 11. <i>wnt8a</i> is identified as a negative regulator of Smad1/5/8 phosphorylation (pSmad1, middle panel) and activator of Wnt signalling (pLRP6, upper panel) at gastrula stage. UI: uninjected.</p

Comparison of fungal cell propagation and antifungal gene expression in insects.

<p><b>A</b>. Comparison of fungal hyphal body (HB) numbers in insect hemocoel 48 hrs post injection. Ten insects were bled for each treatment, and five microscopic fields were observed for each insect. Unpaired <i>t</i>-test was conducted to compare the difference level between WT and mutants. ***, represents the difference level at <i>P</i> < 0.001. <b>B</b>. qRT-PCR analysis of <i>Gal</i> (for gallerimycin) gene expression by insects treated with spores of WT and <i>Blys2</i>-related mutants for 36 hrs. *, <i>P</i> < 0.05. <b>C</b>. qRT-PCR analysis of <i>Gal</i> gene expression by insects treated with spores of WT and <i>Blys5</i>-related mutants for 36 hrs. *, <i>P</i> < 0.05. The <i>Slp1</i> gene of <i>M</i>. <i>oryzae</i> was used to complement the null mutants of <i>Blys2</i> (Δ<i>Blys2</i>::<i>Slp1</i>) and <i>Blys5</i> (Δ<i>Blys5</i>::<i>Slp1</i>), respectively. Overexpress of <i>Blys2</i> in the WT strain of <i>B</i>. <i>bassiana</i> was performed by using two different constitutive promoters, i.e., the <i>GpdA</i> (to obtain the transformant WT::<i>gp-Blys2</i>) and laccase gene (to obtain the transformant WT::<i>lp-Blys2</i>) promoters.</p

Insect hemocyte encapsulation and fungal developments in insect hemocoels on a time scale.

<p>The last instar larvae of wax moth were injected with spores of WT and mutants and bled at different times (labeled on the left) for microscopic examination of insect cellular immune responses and fungal developments. Bar: 10 μm. Arrows in different panels point to fungal cells. For overexpression of <i>Blys2</i> in the WT strains of <i>B</i>. <i>bassiana</i>, both the <i>GpdA</i> (to obtain the transformant WT::<i>gp-Blys2</i>) and laccase (WT::<i>lp-Blys2</i>) gene promoters were used to control gene transcription.</p

Classification of the positive clones identified in the screen.

<p>Values were given as percentages against total clone numbers identified in the screen (<i>n</i> = 20).</p>a<p>Percentage of clones having at least one publication describing their functions in <i>Xenopus</i>.</p>b<p>6 functional groups were established as described.</p>c<p>A total of 20 positive clones have been identified. Clones that modulate the activities of more than one signalling pathways, are counted in each group.</p