Biological and molecular characterization of the response of tomato plants treated with Trichoderma koningiopsis

Tomato-Fusarium oxysporum f.sp. radicis-lycopersici pathosystem was used to study induced systemic resistance elicited by Trichoderma koningiopsis (Th003) using the split root model. The ability of the antagonist to promote plant growth was also established. Stem colonization by the pathogen was significantly reduced in treated plants. The induction of resistance was enhanced 6 days after elicitation and when the antagonist was used in a concentration of 10⁵ conidia per ml. Th003 application in seed priming and nursery significantly stimulated plant growth. Gene expression induced by Th003 was evaluated using the tomato TOM1 microarray. Plant treatment with T. koningiopsis affected mRNA levels of 45 genes: 41 in roots and 4 in leaves. Of particular interest was the induction of genes involved in the jasmonic and ethylene transduction pathways found in the microarray analysis and qRT-PCR, which suggest a temporary increment of defense related gene expression response to T. koningiopsis Th003.     

MALDI-Qq-TOF-MS and transient gene expression analysis indicated co-enhancement of β-1,3-glucanase and endochitinase by tMEK2 and the involvement of divergent pathways

A mitogen-activated protein kinase (MAPK) pathway has been demonstrated as a key pathway in plant defense against pathogen attacks. With proteomics approaches, we specifically studied activation events downstream of a MAPK kinase, tMEK2, in tomato. Overexpression of a constitutively activated tomato MAPK kinase gene (tMEK2^MUT) enhanced resistance of transgenic tomato lines to the virulent bacterial pathogen Pseudomonas syringae pv. tomato. Pathogenesis-related genes, PR1b1, β-1,3-glucanase, and endochitinase were up-regulated by tMEK2^MUT. Two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation-time-of-flight-mass spectrometry analysis of total soluble leaf proteins indicated that β-1,3-glucanase and endochitinase are among the up-regulated proteins in these transgenic plants. Co-expression studies using a transient gene expression system have indicated that β-1,3-glucanase and endochitinase genes up-regulated by tMEK2^MUT were down-regulated by different specific phosphatases through dephosphorylation of certain downstream signaling molecules. Our observations indicate that increased products of β-1,3-glucanase and endochitinase genes downstream of tMEK2 may play an important role in achieving disease resistance.     

Rice <Emphasis Type="Italic">OsPBL1</Emphasis> (ORYZA SATIVA ARABIDOPSIS PBS1-LIKE 1) enhanced defense of <Emphasis Type="Italic">Arabidopsis</Emphasis> against <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> DC3000

The receptor-like cytoplasmic kinases (RLCK family VII) are required for plant defense against various pathogens. Previously, OsPBL1 (ORYZA SATIVA ARABIDOPSIS PBS1-LIKE 1) was isolated from rice as a potential RSV (rice stripe virus) resistant factor, but its physiological roles in plant defense are yet to be investigated. In this study, we demonstrated that OsPBL1increased defense against P. syringae in transgenic Arabidopsis. To ascertain the role of OsPBL1 gene in plant defense, OsPBL1 tagged with HA (i.e. Hemagglutinin) was overexpressed in Arabidopsis and examined for the resistance against Pseudomonas syringae pv. tomato DC3000 (i.e. Pst DC3000). At 3 dpi of Pst DC3000, transgenic Arabidopsis lines exhibited the reduced chlorotic lesion and propagation of P. syringae, compared to wild type. Elevated pathogen resistance of transgenic lines was correlated with increased H₂O₂ accumulation and callose deposition on the infected leaves. It was also revealed that expression levels of salicylic acid dependent genes such as PR1, PR2, and PR5, were induced higher in transgenic lines than wild type. Taken together, our data suggested that OsPBL1 exerted the role in defense against pathogen attacks in plant via mainly facilitating salicylic acid dependent pathway.     

Identification of differentially expressed genes in the mutualistic association of tall fescue with Neotyphodium coenophialum

Tall fescue (Lolium arundinaceum), an agronomically important forage grass, is typically associated with a mutualistic asexual fungus Neotyphodium coenophialum. Plant colonization is endophytic with no symptoms, and fungal growth is confined to the intercellular spaces. The endophyte enhances host fitness by providing protection from various abiotic and biotic stresses and by improving nutrient acquisition. By suppression subtractive hybridization (SSH) we identified 29 genes that are up-regulated or down-regulated in endophyte-infected tall fescue as compared to endophyte-free tall fescue. Of the genes that had matches to known genes present in the NCBI databases (approximately 50%), several had roles related to plant defense and stress tolerance. Differential expression of these genes was confirmed by semi-quantitative RT-PCR, competitive RT-PCR, and northern hybridization. Endophyte-associated changes in gene expression patterns were consistent among cultivars of tall fescue but differed in some other grass–endophyte associations. Our results indicate that both partners in this symbiosis are active participants, and that the endophyte may be suppressing at least one plant defense gene (putatively encoding PR-10). Further analyses of the differentially expressed genes should aid in understanding the fundamental nature of this mutualistic symbiosis and provide insight into the mechanisms of documented endophyte-enhanced plant improvements.     

<Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> upregulates marker genes of the salicylic acid and ethylene signaling pathways but not those of the jasmonic acid pathway in leaflets of <Emphasis Type="Italic">Solanum</Emphasis> lines during early stage of infection

Real-Time PCR assay was used to quantify the expression of marker genes of the salicylic acid, jasmonic acid and ethylene signaling pathways in seven Solanum lines after inoculation with a Ralstonia solanacearum phylotype I strain, R008. Four Solanum lycopersicum lines (CRA 66, Hawaii 7996, MST 32/1, Quatre carrées), one S. tuberosum line (Spunta), the wild Lycopersicon cerasiforme and Solanum commersonii were used for this investigation. Results revealed very little activation of the jasmonic acid pathway marker genes, lipoxygenase A (LoxA) and protease inhibitor II (Pin2), with no significant difference (p > 0.05) in fold change expression among the Solanum lines. In contrast the salicylic acid pathway marker genes, glucanase A (GluA) and PR-1a, and the ethylene pathway marker genes, osmotin-like (Osm) and PR-1b, were expressed at higher levels with a statistically significant difference (p < 0.05) in fold change expression among the Solanum lines. The resistant lines L. cerasiforme, CRA 66, Hawaii 7996 and S. commersonii showed stronger activation of the salicylic acid and ethylene marker genes than the moderately resistant cultivar (MST 32/1) and the susceptible lines (Quatre carrées and Spunta). The marker genes reached their highest expression levels earlier (4 h.p.i) in the resistant and moderately resistant lines than in the susceptible lines (48 h.p.i.). These results indicate that salicylic acid and ethylene signaling pathways have a significant role in defense against R. solanacearum. The timing and magnitude of the upregulation of gene expression may determine the plant ability to put up a defense response against the pathogen.     

Chitin elicitor receptor kinase 1 (CERK1) is required for the non-host defense response of <Emphasis Type="Italic">Arabidopsis</Emphasis> to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. Sp. <Emphasis Type="Italic">cubense</Emphasis>

Banana wilt disease is a typical vascular disease caused by the fungal pathogen Fusarium oxysporum f. sp. cubense 4 (Foc 4). Pattern recognition receptors in the plant cell membrane can recognize pathogen-associated molecular patterns (PAMPs) to activate multi-layer defense responses, including defense gene expression, stomatal closure, reactive oxygen species (ROS) burst and callose deposition, to limit pathogen growth. In the present study, we found that chitin elicitor receptor kinase 1 (CERK1) was required for the non-host resistance of Arabidopsis thaliana to Foc B2 (a strain of Foc 4). The cerk1 mutant had weaker defense responses after Foc B2 treatment, including lower expression of PAMP- and salicylic acid-responsive genes, no stomatal closure, lower ROS level and less callose deposition, than that of the wild-type plant. Consistent with this, the cerk1 mutant plants exhibited higher susceptibility to non-host pathogen Foc B2. These results suggest the crucial importance of CERK1 in Foc B2-triggered non-host resistance.     

Molecular mechanisms of chitin recognition and immune signaling by LysM-receptors

Plants have the ability to recognize microbe-associated molecular patterns (MAMPs) and initiate various defense responses. Chitin is a representative fungal MAMP that triggers defense signaling in a wide range of plant species. In rice, OsCEBiP and OsCERK1 form a receptor complex and play critical roles in chitin-triggered defense signaling. Recently, we found the formation of a unique sandwich-type dimer of OsCEBiP plays an important role for activation of chitin signaling. We now understand why N-acetyl groups and the longer chitin-oligosaccharides are required for receptor binding. We also found OsCERK1 is a bifunctional molecule acting in defense and also in AM symbiosis.     

Genome-wide identification of mildew resistance locus O (<Emphasis Type="Italic">MLO</Emphasis>) genes in tree model poplar (<Emphasis Type="Italic">Populus trichocarpa</Emphasis>): powdery mildew management in woody plants

Poplars are economically important fast growing trees. They are exposed to broad range of fungal diseases like powdery mildew (PM). MLOs (mildew resistance locus O), as plant susceptibility genes, act as negative regulators and whose loss-of-functions confer complete resistance to PM disease. Herein, work identified the MLO gene family members in poplar, a woody model species. A total of 26 identified MLOs (annotated as PtMLO1–26) were distributed on 14 poplar chromosomes either individually or in groups of two to four. PtMLO genes encoded a polypeptide of 341–593 residues with a characteristic MLO domain structure. One tandem and eight segmental duplications were revealed in PtMLO genes. PtMLO proteins anchored at plasma membrane and had putative 5–9 TMDs with extracellular/cytosolic N- and C-terminuses. They were rich in leucine (9.1–12.9%), which is reported to play roles in defense response signaling. The C-terminal calmodulin-binding domain (CaMBD), reported to modulate the signaling mechanism in the defense response, was completely preserved in all PtMLOs, except PtMLO6. This domain was partially absent in PtMLO6 which is inferred to be a different MLO type or a pseudogene with a lost/impaired function in PM response. Besides, second and third cytoplasmic loops that are critical for PM-susceptibility were identified in PtMLOs. Particularly, PtMLO17, 18, 19, and 24 genes, inferred from Arabidopsis-poplar comparative phylogeny, were identified as potential candidates that may be involved in poplar-PM resistance. Notably, inductions of 14 PtMLO genes were detected in probes of microarray data such as GSE56865, GSE16417, and GSE23726 under different fungal infections indicating their involvements in plant defense. Overall, this work provided a basis for woody plant genomics for the effective and better management of poplar-PM disease.     

Agrobacterium-mediated transformation efficiency is altered in a novel rice bacterial blight resistance cultivar and is influenced by environmental temperature

Y73 is a progeny of asymmetric somatic hybridization from an elite japonica rice cultivar (Dalixiang) and a wild rice (Oryza meyeriana), which shows high resistance to bacterial blight. It has a similar genetic background to its recurrent parent, Dalixiang, but Y73 has a high resistance to both bacterial blight and Agrobacterium. The transformation efficiency of Y73 was 35.7%, while Dalixiang had higher transformation efficiency (71.2%) under the same co-cultivation temperature (25 °C). These results indicate that the resistance to Agrobacterium tumefaciens in Y73 was linked with its characteristic of bacterial blight resistance, and was obtained from its donor parent, O. meyeriana. Further studies also showed that the resistance to A. tumefaciens was weakened at a lower temperature (20 °C). To study its molecular mechanism, the expression levels of genes associated with Agrobacterium-mediated transformation (OsMPKs, OsVIP1s and OsPR1s) in rice were investigated by Quantitative real-time PCR (qRT-PCR) analysis. The expression levels of OsMPK genes remained unchanged at different temperatures, while all OsVIP1s and almost all OsPR1s were up-regulated and transformation efficiency of Y73 was increased notably when infected by Agrobacterium at 20 °C compared with 25 °C. This study suggests that the bacterial blight resistance genes in Y73 also have an effect on Agrobacterium-mediated transformation and that the response to temperature is associated with the regulation of MAPK/VIP1 defense signaling pathway.     

Development of the infection strategy of the hemibiotrophic plant pathogen,Colletotrichum orbiculare,and plant immunity

The hemibiotrophic fungus Colletotrichum orbiculare forms appressoria as infection structures and primarily establishes biotrophic infection in cucumber epidermal cells. Subsequently, it develops necrotrophic infection. In the pre-invasion stage, morphogenesis of appressoria of C. orbiculare is triggered by signals from the plant surface. We found that C. orbiculare PAG1 (Perish-in-the-Absence-of-GYP1), a component of MOR [morphogenesis-related NDR (nuclear Dbf2-related) kinase network] plays an essential role as a key component of the plant-specific signaling pathway and that hydrolysis of cutin by a spore surface esterase creates a cutin monomer that constitutes a key plant-derived signal. Development of the infection structure of C. orbiculare is strictly regulated by the cell cycle and we found that proper regulation of G1/S progression via two-component GAP genes, consisting of BUB2 (Budding-Uninhibited-by-Benomyl-2) and BFA1 (Byr-Four-Alike-1) is essential for the establishment of successful infection. In the post-invasion stage, the establishment of the biotrophic phase of hemibiotrophic fungi is crucial for successful infection. We found that C. orbiculare WHI2 (WHIsky-2), an Saccharomyces cerevisiae stress regulator homolog, is involved in the phase transition from biotrophy to necrotrophy through TOR (Target of Rapamycin) signaling, and is thus essential for full pathogenesis.     

Volatile 1-octen-3-ol induces a defensive response in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

1-Octen-3-ol is a major volatile metabolite produced by mold fungi. When Arabidopsis plants were treated with 1-octen-3-ol, some defense genes that are turned on by wounding or ethylene/jasmonic acid signaling were induced. The treatment also enhanced resistance of the plant against Botrytis cinerea. When the induction of defense genes with 1-octen-3-ol was compared with that by volatile methyl jasmonate (MeJA) and methyl salicylate treatments, the induction pattern was similar to that caused by MeJA. Thus, Arabidopsis seems to recognize 1-octen-3-ol and consequently activates its defense response.