An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil

The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current ...

Nanjing Agricultural University

Pseudomonas fluorescens strain 3JW1, which has a broad-spectrum antimicrobial activity, was studied to investigate whether it affects the amounts of aflatoxin B1 (AFB1) produced by Aspergillus flavus. It was found that the bacterium reduced the amounts of AFB1 in potato dextrose broth (PDB) and peanut medium by 97.8% and 99.4%, respectively. It also reduced AFB1 by ~183 μg/kg (55.8%) when applied onto peanut kernels. This strain reduced AFB1 via three mechanisms. First, it significantly inhibited A. flavus growth; second, our data showed that strain 3JW1 inhibits aflatoxin biosynthesis by A. flavus; and third, P. fluorescens strain 3JW1 is capable of degrading AFB1 at a rate as high as 88.3% in 96 hours. This is the first report demonstrating that Pseudomonas fluorescens can reduce toxin contamination caused by A. flavus on peanut kernels. Our findings indicate that P. fluorescens strain 3JW1 had multiple effects including reducing A. flavus infection and aflatoxin contamination. And the results also highlight the potential applications of the strain 3JW1 for the biological control of aflatoxin contamination in peanuts and other susceptible crops.

Investigation of Pseudomonas fluorescens strain 3JW1 on preventing and reducing aflatoxin contaminations in peanuts.

Sucrose non-fermenting-1-related protein kinase 1 (SnRK1) has been shown to play an essential role in regulating saccharide metabolism and starch biosynthesis of plant. The regulatory role of  from potato in regulating carbohydrate metabolism and starch accumulation has not been investigated. In this work, a cDNA encoding the SnRK1 protein, named , was isolated from potato. The open reading frame contained 1545 nucleotides encoding 514 amino acids. Subcellular localization analysis in onion epidermal cells indicated that StSnRK1 protein was localized to the nucleus. The coding region of  was cloned into a binary vector under the control of 35S promoter and then transformed into tobacco to obtain transgenic plants. Transgenic tobacco plants expressing  were shown to have a significant increased accumulation of starch content, as well as sucrose, glucose and fructose content. Real-time quantitative PCR analysis indicated that overexpression of  up-regulated the expression of sucrose synthase (), ADP-glucose pyrophosphorylase (AGPase) and soluble starch synthase () genes involved in starch biosynthesis in the transgenic plants. In contrast, the expression of sucrose phosphate synthase () gene was decreased in the transgenic plants. Meanwhile, enzymatic analyses indicated that the activities of major enzymes (SUS, AGPase and SSS) involved in the starch biosynthesis were enhanced, whereas SPS activity was decreased in the transgenic plants compared to the wild-type. These results suggest that the manipulation of  expression might be used for improving quality of plants in the future.

A sucrose non-fermenting-1-related protein kinase 1 gene from potato, , regulates carbohydrate metabolism in transgenic tobacco.

Soybean contains a group of 64 L-type lectin receptor-like kinases. Three LecRKs were involved in the interactions with Phytophthora sojae and Bradyrhizobium diazoefficiens. L-type lectin receptor-like kinases (LecRKs) comprise an important class of membrane-localized receptor-like kinases that are involved in plant adaptation. In this study, we performed an inventory analysis of LecRKs in Glycine max (soybean). In total, 64 GmLecRKs containing the canonical LecRK feature were identified. Phylogenetic analysis revealed that 48 GmLecRKs have close orthologs in Arabidopsis or Solanum lycopersicum, while 16 are likely present only in the leguminous plant species. Transcriptome analyses revealed that expressions of multiple GmLecRK genes are either induced or suppressed during infection by the soybean root rot pathogen Phytophthora sojae. In addition, overexpression of the three LecRKs (Glyma.17G085000, Glyma.05G041300 or Glyma.17G224600) in the soybean hairy roots enhanced resistance to P. sojae. Upon inoculation with Bradyrhizobium diazoefficiens, overexpression of Glyma.17G085000 in the soybean hairy roots does not significantly influence the nodulation, while overexpression of Glyma.05G041300 or Glyma.17G224600 slightly reduced the number and dry weight of nodules. This study highlights the importance of LecRKs in regulating plant-microbe interactions and provides new knowledge on the deployment of LecRKs to increase resistance in soybean.

Identification and characterization of L-type lectin receptor-like kinases involved in Glycine max-Phytophthora sojae interaction.

 (Ds) and  (Ff; previously called ) are two closely related Dothideomycete fungal species that cause Dothistroma needle blight in pine and leaf mold in tomato, respectively. During host colonization, these pathogens secrete virulence factors termed effectors to promote infection. In the presence of corresponding host immune receptors, however, these effectors activate plant defenses, including a localized cell death response that halts pathogen growth. We identified two apoplastic effector protein families, Ecp20 and Ecp32, which are conserved between the two pathogens. The Ecp20 family has four paralogues in both species, while the Ecp32 family has four paralogues in  and five in . Both families have members that are highly expressed during host infection. Members of the Ecp20 family have predicted structural similarity to proteins with a β-barrel fold, including the Alt a 1 allergen from , while members of the Ecp32 family have predicted structural similarity to proteins with a β-trefoil fold, such as trypsin inhibitors and lectins. Using -mediated transient transformation assays, each family member was assessed for its ability to trigger cell death in leaves of the non-host species  and . Using this approach, FfEcp20-2, DsEcp20-3, and FfEcp20-3 from the Ecp20 family, and all members from the Ecp32 family, except for the Ds/FfEcp32-4 pair, triggered cell death in both species. This cell death was dependent on secretion of the effectors to the apoplast. In line with recognition by an extracellular immune receptor, cell death triggered by Ds/FfEcp20-3 and FfEcp32-3 was compromised in  silenced for  or , which encode extracellular co-receptors involved in transducing defense response signals following apoplastic effector recognition. We then investigated whether DsEcp20-3 and DsEcp20-4 triggered cell death in the host species  by directly infiltrating purified protein into pine needles. Strikingly, as in the non-host species, DsEcp20-3 triggered cell death, while DsEcp20-4 did not. Collectively, our study describes two new candidate effector families with cell death-eliciting activity from  and  and provides evidence that members of these families are recognized by plant immune receptors.

Characterization of two conserved cell death elicitor families from the Dothideomycete fungal pathogens  and  (syn. ).

Plants have evolved sophisticated immune networks to restrict pathogen colonization. In response, pathogens deploy numerous virulent effectors to circumvent plant immune responses. However, the molecular mechanisms by which pathogen-derived effectors suppress plant defenses remain elusive. Here, we report that the nucleus-localized RxLR effector PsAvh110 from the pathogen Phytophthora sojae, causing soybean (Glycine max) stem and root rot, modulates the activity of a transcriptional complex to suppress plant immunity. Soybean like-heterochromatin protein 1-2 (GmLHP1-2) and plant homeodomain finger protein 6 (GmPHD6) form a transcriptional complex with transcriptional activity that positively regulates plant immunity against Phytophthora infection. To suppress plant immunity, the nuclear effector PsAvh110 disrupts the assembly of the GmLHP1-2/GmPHD6 complex via specifically binding to GmLHP1-2, thus blocking its transcriptional activity. We further show that PsAvh110 represses the expression of a subset of immune-associated genes, including BRI1-associated receptor kinase 1-3 (GmBAK1-3) and pathogenesis-related protein 1 (GmPR1), via G-rich elements in gene promoters. Importantly, PsAvh110 is a conserved effector in different Phytophthora species, suggesting that the PsAvh110 regulatory mechanism might be widely utilized in the genus to manipulate plant immunity. Thus, our study reveals a regulatory mechanism by which pathogen effectors target a transcriptional complex to reprogram transcription.

The Phytophthora sojae nuclear effector PsAvh110 targets a host transcriptional complex to modulate plant immunity.

Elicitins are a large family of secreted proteins in Phytophthora. Clade 1 elicitins were identified decades ago as potent elicitors of immune responses in Nicotiana species, but the mechanisms underlying elicitin recognition are largely unknown. Here we identified an elicitin receptor in Nicotiana benthamiana that we named REL for Responsive to ELicitins. REL is a receptor-like protein (RLP) with an extracellular leucine-rich repeat (LRR) domain that mediates Phytophthora resistance by binding elicitins. Silencing or knocking out REL in N. benthamiana abolished elicitin-triggered cell death and immune responses. Domain deletion and site-directed mutagenesis revealed that the island domain (ID) located within the LRR domain of REL is crucial for elicitin recognition. In addition, sequence polymorphism in the ID underpins the genetic diversity of REL homologs in various Nicotiana species in elicitin recognition and binding. Remarkably, REL is phylogenetically distant from the elicitin response (ELR) protein, an LRR-RLP that was previously identified in the wild potato species Solanum microdontum and REL and ELR differ in the way they bind and recognize elicitins. Our findings provide insights into the molecular basis of plant innate immunity and highlight a convergent evolution of immune receptors towards perceiving the same elicitor.

Convergent evolution of immune receptors underpins distinct elicitin recognition in closely related Solanaceous plants.

The detection of microbial infections by plants induces the rapid formation of immune receptor complexes at the plasma membrane. However, how this process is controlled to ensure proper immune signaling remains largely unknown. Here, we found that the Nicotiana benthamiana membrane-localized leucine-rich repeat receptor-like kinase BAK1-INTERACTING RLK 2 (NbBIR2) constitutively associates with BRI1-ASSOCIATED RECEPTOR KINASE 1 (NbBAK1) in vivo and in vitro and promotes complex formation with pattern recognition receptors. In addition, NbBIR2 is targeted by 2 RING-type ubiquitin E3 ligases, SNC1-INFLUENCING PLANT E3 LIGASE REVERSE 2a (NbSNIPER2a) and NbSNIPER2b, for ubiquitination and subsequent degradation in planta. NbSNIPER2a and NbSNIPER2b interact with NbBIR2 in vivo and in vitro and are released from NbBIR2 upon treatment with different microbial patterns. Furthermore, accumulation of NbBIR2 in response to microbial patterns is tightly associated with NbBAK1 abundance in N. benthamiana. NbBAK1 acts as a modular protein that stabilizes NbBIR2 by competing with NbSNIPER2a or NbSNIPER2b for association with NbBIR2. Similar to NbBAK1, NbBIR2 positively regulates pattern-triggered immunity and resistance to bacterial and oomycete pathogens in N. benthamiana, whereas NbSNIPER2a and NbSNIPER2b have the opposite effect. Together, these results reveal a feedback regulatory mechanism employed by plants to tailor pattern-triggered immune signaling.

Zhiyuan Chen

Nanjing Agricultural University

An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil