Trehalose Biosynthesis Promotes Pseudomonas aeruginosa Pathogenicity in Plants

Pseudomonas aeruginosa strain PA14 is a multi-host pathogen that infects plants, nematodes, insects, and vertebrates. Many PA14 factors are required for virulence in more than one of these hosts. Noting that plants have a fundamentally different cellular architecture from animals, we sought to identify PA14 factors that are specifically required for plant pathogenesis. We show that synthesis by PA14 of the disaccharide trehalose is required for pathogenesis in Arabidopsis, but not in nematodes, insects, or mice. In-frame deletion of two closely-linked predicted trehalose biosynthetic operons, treYZ and treS, decreased growth in Arabidopsis leaves about 50 fold. Exogenously co-inoculated trehalose, ammonium, or nitrate, but not glucose, sulfate, or phosphate suppressed the phenotype of the double ΔtreYZΔtreS mutant. Exogenous trehalose or ammonium nitrate does not suppress the growth defect of the double ΔtreYZΔtreS mutant by suppressing the plant defense response. Trehalose also does not function intracellularly in P. aeruginosa to ameliorate a variety of stresses, but most likely functions extracellularly, because wild-type PA14 rescued the in vivo growth defect of the ΔtreYZΔtreS in trans. Surprisingly, the growth defect of the double ΔtreYZΔtreS double mutant was suppressed by various Arabidopsis cell wall mutants that affect xyloglucan synthesis, including an xxt1xxt2 double mutant that completely lacks xyloglucan, even though xyloglucan mutants are not more susceptible to pathogens and respond like wild-type plants to immune elicitors. An explanation of our data is that trehalose functions to promote the acquisition of nitrogen-containing nutrients in a process that involves the xyloglucan component of the plant cell wall, thereby allowing P. aeruginosa to replicate in the intercellular spaces in a leaf. This work shows how P. aeruginosa, a multi-host opportunistic pathogen, has repurposed a highly conserved “house-keeping” anabolic pathway (trehalose biosynthesis) as a potent virulence factor that allows it to replicate in the intercellular environment of a leaf

Tvbgn3, a β-1,6-Glucanase from the Biocontrol Fungus Trichoderma virens, Is Involved in Mycoparasitism and Control of Pythium ultimum

Even though β-1,6-glucanases have been purified from several filamentous fungi, the physiological function has not been conclusively established for any species. In the present study, the role of Tvbgn3, a β-1,6-glucanase from Trichoderma virens, was examined by comparison of wild-type (WT) and transformant strains in which Tvbgn3 was disrupted (GKO) or constitutively overexpressed (GOE). Gene expression analysis revealed induction of Tvbgn3 in the presence of host fungal cell walls, indicating regulation during mycoparasitism. Indeed, while deletion or overexpression of Tvbgn3 had no evident effect on growth and development, GOE and GKO strains showed an enhanced or reduced ability, respectively, to inhibit the growth of the plant pathogen Pythium ultimum compared to results with the WT. The relevance of this activity in the biocontrol ability of T. virens was confirmed in plant bioassays. Deletion of the gene resulted in levels of disease protection that were significantly reduced from WT levels, while GOE strains showed a significantly increased biocontrol capability. These results demonstrate the involvement of β-1,6-glucanase in mycoparasitism and its relevance in the biocontrol activity of T. virens, opening a new avenue for biotechnological applications

A Proteinaceous Elicitor Sm1 from the Beneficial Fungus Trichoderma virens Is Required for Induced Systemic Resistance in Maize1[W]

We have previously shown that the beneficial filamentous fungus Trichoderma virens secretes the highly effective hydrophobin-like elicitor Sm1 that induces systemic disease resistance in the dicot cotton (Gossypium hirsutum). In this study we tested whether colonization of roots by T. virens can induce systemic protection against a foliar pathogen in the monocot maize (Zea mays), and we further demonstrated the importance of Sm1 during maize-fungal interactions using a functional genomics approach. Maize seedlings were inoculated with T. virens Gv29-8 wild type and transformants in which SM1 was disrupted or constitutively overexpressed in a hydroponic system or in soil-grown maize seedlings challenged with the pathogen Colletotrichum graminicola. We show that similar to dicot plants, colonization of maize roots by T. virens induces systemic protection of the leaves inoculated with C. graminicola. This protection was associated with notable induction of jasmonic acid- and green leaf volatile-biosynthetic genes. Neither deletion nor overexpression of SM1 affected normal growth or development of T. virens, conidial germination, production of gliotoxin, hyphal coiling, hydrophobicity, or the ability to colonize maize roots. Plant bioassays showed that maize grown with SM1-deletion strains exhibited the same levels of systemic protection as non-Trichoderma-treated plants. Moreover, deletion and overexpression of SM1 resulted in significantly reduced and enhanced levels of disease protection, respectively, compared to the wild type. These data together indicate that T. virens is able to effectively activate systemic disease protection in maize and that the functional Sm1 elicitor is required for this activity

Pathogen-Secreted Proteases Activate a Novel Plant Immune Pathway

Mitogen-Activated Protein Kinase (MAPK) cascades play central roles in innate immune signaling networks in plants and animals1,2. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive1. We report that pathogen-secreted proteases activate a previously unknown signaling pathway in Arabidopsis thaliana involving the Gα, Gβ and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of a MAPK cascade. In this pathway, Receptor for Activated C Kinase 1 (RACK1) functions as a novel scaffold that binds to the Gβ subunit as well as to all three tiers of the MAPK cascade, thereby linking upstream G protein signaling to downstream activation of a MAPK cascade. The protease-G protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signaling pathways such as the one elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to a MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the novel protease-mediated immune signaling pathway described here was facilitated by the use of the broad host range, opportunistic bacterial pathogen Pseudomonas aeruginosa. The ability of P. aeruginosa to infect both plants and animals makes it an excellent model to identify novel types of immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems

Trehalose levels in the trehalose mutants.

<p><i>P. aeruginosa</i> strains were grown at 37°C in MinA medium supplemented with 0.5 M NaCl. Trehalose was extracted and quantified enzymatically as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003217#s4" target="_blank">Materials and Methods</a>. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003217#ppat-1003217-g001" target="_blank">Figure 1</a> for a description of the mutants. Data represent the mean ± SE of two replicate samples and are representative of at least three independent experiments.</p

PA14 trehalose mutants are rescued in trans <i>in planta</i> by wild-type PA14.

<p>(A) Four-week-old Arabidopsis Col-0 plants were inoculated with a 1∶1 mixture of PA14 wild-type carrying pSMC2 (GFP) and the <i>ΔtreYZΔtreS</i> mutant carrying pAA100 (DsRed) at a total concentration of 3.5×10² CFU/cm² leaf area. As a control the strains were inoculated individually. (B) In the left panel, four-week-old Arabidopsis Col-0 plants were inoculated with a 1∶1 mixture of PA14 wild-type carrying pSMC2 (GFP) and the Δ<i>42</i> mutant carrying pAA100 (DsRed) at a total concentration of 6×10² CFU/cm² leaf area. As a control the strains were inoculated individually. In the right panel, PA14 wild-type carried pAA100 and the Δ<i>42</i> mutant carried pSMC2. Three days post infiltration, leaves were harvested and CFU determined by counting colonies using a Zeiss Stemi SV6 dissecting microscope fitted with a dual GFP/RFP filter. Data represent the mean of bacterial titers ± SE of six leaf disks excised from 6 leaves of 3 plants and are representative of three independent experiments.</p

The <i>ΔtreYZΔtreS</i> mutant is more virulent than wild-type PA14 in nematodes and mice.

<p>(A) <i>C. elegans</i> are more susceptible to killing by <i>ΔtreYZΔtreS</i> than PA14 wild-type (P<0.0001). Mutant <i>fer15;fem1 C. elegans</i> animals were exposed to <i>P. aeruginosa</i> strains and survival was determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003217#s4" target="_blank">Materials and Methods</a>. Data at each time point correspond to the average of three plates per strain, each with approximately 40 animals per plate, and are representative of two independent experiments. (B) The Δ<i>42</i> mutant is more virulent than wild-type PA14 in a murine acute lung infection model. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003217#s4" target="_blank">Materials and Methods</a> for details of infection protocol. The median CFU/gram of lung tissue of mice infected with <i>ΔtreYZΔtreS</i> is 2-fold higher than with wild-type PA14 18 hours post intranasal infection (P<0.05, Mann-Whitney U test). Data are representative of two independent experiments.</p

The <i>in planta</i> growth defect of the PA14 <i>ΔtreYZΔtreS</i> mutant in Arabidopsis is suppressed by cell wall mutations.

<p>Growth of PA14 wild-type and the <i>ΔtreYZΔtreS</i> mutant 3 days post infiltration in Arabidopsis cell wall mutants <i>mur2-1</i>, <i>mur3-2</i>, <i>mur4-1</i>, <i>mur10-2</i> and <i>xxt1/xxt2</i>. Data represent the mean of bacterial titers ± SE of six leaf disks excised from 6 leaves of 3 plants. Letters above bars denote statistically significant differences (P<0.05, Fisher's PLSD test). The experiments were repeated at least two times.</p

The Δ<i>42</i> mutant is not more susceptible to osmotic or oxidative stress.

<p>(A) Growth of Δ<i>42, ΔNAGGN</i> and PA14 wild-type under osmotic stress <i>in vitro</i>. Cells were grown at 37°C in MinA medium supplemented with 0.5 M NaCl. Data represent the mean ± SE of 3 replicates. (B) The <i>in planta</i> growth defect of Δ<i>42</i> is suppressed by trehalose but not betaine. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003217#ppat-1003217-g002" target="_blank">Figure 2</a> for experimental details. Data represent the mean ± SE of six replicate samples. (C) <i>In vitro</i> survival of PA14 wild-type, Δ<i>42</i>, and a PA14 <i>zwf</i>::<i>MAR2xT7</i> mutant cultured for three days in MinA medium supplemented with various concentrations of paraquat (PQ). (D) Survival of PA14 wild-type and Δ<i>42</i> in LB medium containing 1 M or 2 M hydrogen peroxide added directly to overnight cultures grown for 14 h (inoculum, zero time point on x axis). Cells were further incubated for 8 h at 37°C. 3 M H₂O₂ was a lethal dose. All experiments in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003217#ppat-1003217-g008" target="_blank">Figure 8</a> were repeated at least two times.</p

Arabidopsis cell wall mutants are not more susceptible to virulent or non-pathogenic <i>P. syringae</i> strains and the <i>xxt1/xxt2</i> mutant mounts an effective innate immune response.

<p>Growth of (A) <i>P. syringae</i> pv. <i>tomato</i> strain DC3000 (B) <i>P. syringae</i> pv. <i>tomato</i> strain DC3000 <i>hrcC</i> (C) <i>P. syringae</i> pv. <i>phaseolicola</i> strain 3121 three days post infiltration in Arabidopsis cell wall mutants <i>mur2-1</i>, <i>mur3-2</i>, <i>mur4-1</i>, <i>mur10-2</i> and <i>xxt1/xxt2</i> and Col-0 wild-type and (D) <i>P. syringae</i> pv. <i>tomato</i> strain DC3000 three days post infiltration of Col-0 and <i>xxt1/xxt2</i> mutant preinfiltrated with 1 µM flg22 for 24 hours. Data represent the mean of bacterial titers ± SE of six leaf disks excised from 6 leaves of 3 plants. Letters above bars denote statistically significant differences (P<0.05, Fisher's PLSD test). Absence of letters indicates no statistically significant differences. The experiments were repeated at least two times.</p