A central circadian oscillator confers defense heterosis in hybrids without growth vigor costs.

Plant immunity frequently incurs growth penalties, which known as the trade-off between immunity and growth. Heterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits but rarely for disease resistance. Here, we report that the central circadian oscillator, CCA1, confers heterosis for bacterial defense in hybrids without growth vigor costs, and it even significantly enhances the growth heterosis of hybrids under pathogen infection. The genetic perturbation of CCA1 abrogated heterosis for both defense and growth in hybrids. Upon pathogen attack, the expression of CCA1 in F1 hybrids is precisely modulated at different time points during the day by its rhythmic histone modifications. Before dawn of the first infection day, epigenetic activation of CCA1 promotes an elevation of salicylic acid accumulation in hybrids, enabling heterosis for defense. During the middle of every infection day, diurnal epigenetic repression of CCA1 leads to rhythmically increased chlorophyll synthesis and starch metabolism in hybrids, effectively eliminating the immunity-growth heterosis trade-offs in hybrids.

Critical assessment and performance improvement of plant-pathogen protein-protein interaction prediction methods.

The identification of plant-pathogen protein-protein interactions (PPIs) is an attractive and challenging research topic for deciphering the complex molecular mechanism of plant immunity and pathogen infection. Considering that the experimental identification of plant-pathogen PPIs is time-consuming and labor-intensive, computational methods are emerging as an important strategy to complement the experimental methods. In this work, we first evaluated the performance of traditional computational methods such as interolog, domain-domain interaction and domain-motif interaction in predicting known plant-pathogen PPIs. Owing to the low sensitivity of the traditional methods, we utilized Random Forest to build an inter-species PPI prediction model based on multiple sequence encodings and novel network attributes in the established plant PPI network. Critical assessment of the features demonstrated that the integration of sequence information and network attributes resulted in significant and robust performance improvement. Additionally, we also discussed the influence of Gene Ontology and gene expression information on the prediction performance. The Web server implementing the integrated prediction method, named InterSPPI, has been made freely available at http://systbio.cau.edu.cn/intersppi/index.php. InterSPPI could achieve a reasonably high accuracy with a precision of 73.8% and a recall of 76.6% in the independent test. To examine the applicability of InterSPPI, we also conducted cross-species and proteome-wide plant-pathogen PPI prediction tests. Taken together, we hope this work can provide a comprehensive understanding of the current status of plant-pathogen PPI predictions, and the proposed InterSPPI can become a useful tool to accelerate the exploration of plant-pathogen interactions.

Arabidopsis phospholipase Dα1 and Dδ oppositely modulate EDS1- and SA-independent basal resistance against adapted powdery mildew.

Plants use a tightly regulated immune system to fight off various pathogens. Phospholipase D (PLD) and its product, phosphatidic acid, have been shown to influence plant immunity; however, the underlying mechanisms remain unclear. Here, we show that the Arabidopsis mutants pldα1 and pldδ, respectively, exhibited enhanced resistance and enhanced susceptibility to both well-adapted and poorly adapted powdery mildew pathogens, and a virulent oomycete pathogen, indicating that PLDα1 negatively while PLDδ positively modulates post-penetration resistance. The pldα1δ double mutant showed a similar infection phenotype to pldα1, genetically placing PLDα1 downstream of PLDδ. Detailed genetic analyses of pldδ with mutations in genes for salicylic acid (SA) synthesis (SID2) and/or signaling (EDS1 and PAD4), measurement of SA and jasmonic acid (JA) levels, and expression of their respective reporter genes indicate that PLDδ contributes to basal resistance independent of EDS1/PAD4, SA, and JAsignaling. Interestingly, while PLDα1-enhanced green fluorescent protein (eGFP) was mainly found in the tonoplast before and after haustorium invasion, PLDδ-eGFP's focal accumulation to the plasma membrane around the fungal penetration site appeared to be suppressed by adapted powdery mildew. Together, our results demonstrate that PLDα1 and PLDδ oppositely modulate basal, post-penetration resistance against powdery mildew through a non-canonical mechanism that is independent of EDS1/PAD4, SA, and JA.

Chemical priming of immunity without costs to plant growth.

ß-Aminobutyric acid (BABA) induces broad-spectrum disease resistance, but also represses plant growth, which has limited its exploitation in crop protection. BABA perception relies on binding to the aspartyl-tRNA synthetase (AspRS) IBI1, which primes the enzyme for secondary defense activity. This study aimed to identify structural BABA analogues that induce resistance without stunting plant growth. Using site-directed mutagenesis, we demonstrate that the (l)-aspartic acid-binding domain of IBI1 is critical for BABA perception. Based on interaction models of this domain, we screened a small library of structural BABA analogues for growth repression and induced resistance against biotrophic Hyaloperonospora arabidopsidis (Hpa). A range of resistance-inducing compounds were identified, of which (R)-ß-homoserine (RBH) was the most effective. Surprisingly, RBH acted through different pathways than BABA. RBH-induced resistance (RBH-IR) against Hpa functioned independently of salicylic acid, partially relied on camalexin, and was associated with augmented cell wall defense. RBH-IR against necrotrophic Plectosphaerella cucumerina acted via priming of ethylene and jasmonic acid defenses. RBH-IR was also effective in tomato against Botrytis cinerea. Metabolic profiling revealed that RBH, unlike BABA, does not majorly affect plant metabolism. RBH primes distinct defense pathways against biotrophic and necrotrophic pathogens without stunting plant growth, signifying strong potential for exploitation in crop protection.

Rhizosphere-associated Pseudomonas induce systemic resistance to herbivores at the cost of susceptibility to bacterial pathogens.

Plant-associated soil microbes are important mediators of plant defence responses to diverse above-ground pathogen and insect challengers. For example, closely related strains of beneficial rhizosphere Pseudomonas spp. can induce systemic resistance (ISR), systemic susceptibility (ISS) or neither against the bacterial foliar pathogen Pseudomonas syringae pv. tomato DC3000 (Pto DC3000). Using a model system composed of root-associated Pseudomonas spp. strains, the foliar pathogen Pto DC3000 and the herbivore Trichoplusia ni (cabbage looper), we found that rhizosphere-associated Pseudomonas spp. that induce either ISS and ISR against Pto DC3000 all increased resistance to herbivory by T. ni. We found that resistance to T. ni and resistance to Pto DC3000 are quantitative metrics of the jasmonic acid (JA)/salicylic acid (SA) trade-off and distinct strains of rhizosphere-associated Pseudomonas spp. have distinct effects on the JA/SA trade-off. Using genetic analysis and transcriptional profiling, we provide evidence that treatment of Arabidopsis with Pseudomonas sp. CH267, which induces ISS against bacterial pathogens, tips the JA/SA trade-off towards JA-dependent defences against herbivores at the cost of a subset of SA-mediated defences against bacterial pathogens. In contrast, treatment of Arabidopsis with the ISR strain Pseudomonas sp. WCS417 disrupts JA/SA antagonism and simultaneously primes plants for both JA- and SA-mediated defences. Our findings show that ISS against the bacterial foliar pathogens triggered by Pseudomonas sp. CH267, which is a seemingly deleterious phenotype, may in fact be an adaptive consequence of increased resistance to herbivory. Our work shows that pleiotropic effects of microbiome modulation of plant defences are important to consider when using microbes to modify plant traits in agriculture.

The phenotypic and molecular assessment of the non-conserved Arabidopsis MICRORNA163/S-ADENOSYL-METHYLTRANSFERASE regulatory module during biotic stress.

In plants, microRNAs (miRNAs) have evolved in parallel to the protein-coding genes that they target for expression regulation, and miRNA-directed gene expression regulation is central to almost every cellular process. MicroRNA, miR163, is unique to the Arabidopsis genus and is processed into a 24-nucleotide (nt) mature small regulatory RNA (sRNA) from a single precursor transcript transcribed from a single locus, the MIR163 gene. The MIR163 locus is a result of a recent inverted duplication event of one of the five closely related S-ADENOSYL-METHYLTRANSFERASE genes that the mature miR163 sRNA targets for expression regulation. Currently, however, little is known about the role of the miR163/S-ADENOSYL-METHYLTRANSFERASE regulatory module in response to biotic stress. Here, we document the expression domains of MIR163 and the S-ADENOSYL-METHYLTRANSFERASE target genes following fusion of their putative promoter sequences to the ß-glucuronidase (GUS) reporter gene and subsequent in planta expression. Further, we report on our phenotypic and molecular assessment of Arabidopsis thaliana plants with altered miR163 accumulation, namely the mir163-1 and mir163-2 insertion knockout mutants and the miR163 overexpression line, the MIR163-OE plant. Finally, we reveal miR163 accumulation and S-ADENOSYL-METHYLTRANSFERASE target gene expression post treatment with the defence elicitors, salicylic acid and jasmonic acid, and following Fusarium oxysporum infection, wounding, and herbivory attack. Together, the work presented here provides a comprehensive new biological insight into the role played by the Arabidopsis genus-specific miR163/S-ADENOSYL-METHYLTRANSFERASE regulatory module in normal A. thaliana development and during the exposure of A. thaliana plants to biotic stress.

Assessment of Cytokinin-Induced Immunity Through Quantification of Hyaloperonospora arabidopsidis Infection in Arabidopsis thaliana.

Cytokinins have been shown to regulate plant immunity. Application of high levels of cytokinin to plants leads to decreased susceptibility to pathogens. In this chapter, we describe a fast and accurate protocol for assessment of cytokinin-induced immunity in Arabidopsis plants against an oomycete plant pathogen.

Re-purposing bridging flocculation for on-site, rapid, qualitative DNA detection in resource-poor settings.

Developing molecular diagnostics in resource-poor settings is challenging. As such, we purpose-built a novel bridging flocculation assay for qualitative evaluation of isothermally amplified DNA by naked eye. The flocculation assay was dependent on pH, DNA polymer amounts and lengths. The method was first applied to the rapid and sensitive detection of important plant pathogens and subsequently extended to other pathogens across the animal kingdom to demonstrate the wide applications of our approach.

Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors.

Protein networks and signaling cascades are key mechanisms for intra- and intercellular signal transduction. Identifying the interacting partners of a protein can provide vital clues regarding its physiological role. The bimolecular fluorescence complementation (BiFC) assay has become a routine tool for in vivo analysis of protein-protein interactions and their subcellular location. Although the BiFC system has improved since its inception, the available options for in planta analysis are still subject to very low signal-to-noise ratios, and a systematic comparison of BiFC confounding background signals has been lacking. Background signals can obscure weak interactions, provide false positives, and decrease confidence in true positives. To overcome these problems, we performed an extensive in planta analysis of published BiFC fragments used in metazoa and plants, and then developed an optimized single vector BiFC system which utilizes monomeric Venus (mVenus) split at residue 210, and contains an integrated mTurquoise2 marker to precisely identify transformed cells in order to distinguish true negatives. Here we provide our streamlined double ORF expression (pDOE) BiFC system, and show that our advance in BiFC methodology functions even with an internally fused mVenus210 fragment. We illustrate the efficacy of the system by providing direct visualization of Arabidopsis MLO1 interacting with a calmodulin-like (CML) protein, and by showing that heterotrimeric G-protein subunits Gα (GPA1) and Gß (AGB1) interact in plant cells. We further demonstrate that GPA1 and AGB1 each physically interact with PLDα1 in planta, and that mutation of the so-called PLDα1 'DRY' motif abolishes both of these interactions.

A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis.

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.

Functional assessment of the Medicago truncatula NIP/LATD protein demonstrates that it is a high-affinity nitrate transporter.

The Medicago truncatula NIP/LATD (for Numerous Infections and Polyphenolics/Lateral root-organ Defective) gene encodes a protein found in a clade of nitrate transporters within the large NRT1(PTR) family that also encodes transporters of dipeptides and tripeptides, dicarboxylates, auxin, and abscisic acid. Of the NRT1(PTR) members known to transport nitrate, most are low-affinity transporters. Here, we show that M. truncatula nip/latd mutants are more defective in their lateral root responses to nitrate provided at low (250 µm) concentrations than at higher (5 mm) concentrations; however, nitrate uptake experiments showed no discernible differences in uptake in the mutants. Heterologous expression experiments showed that MtNIP/LATD encodes a nitrate transporter: expression in Xenopus laevis oocytes conferred upon the oocytes the ability to take up nitrate from the medium with high affinity, and expression of MtNIP/LATD in an Arabidopsis chl1(nrt1.1) mutant rescued the chlorate susceptibility phenotype. X. laevis oocytes expressing mutant Mtnip-1 and Mtlatd were unable to take up nitrate from the medium, but oocytes expressing the less severe Mtnip-3 allele were proficient in nitrate transport. M. truncatula nip/latd mutants have pleiotropic defects in nodulation and root architecture. Expression of the Arabidopsis NRT1.1 gene in mutant Mtnip-1 roots partially rescued Mtnip-1 for root architecture defects but not for nodulation defects. This suggests that the spectrum of activities inherent in AtNRT1.1 is different from that possessed by MtNIP/LATD, but it could also reflect stability differences of each protein in M. truncatula. Collectively, the data show that MtNIP/LATD is a high-affinity nitrate transporter and suggest that it could have another function.

Assessment of resistance pathways induced in Arabidopsis thaliana by hypovirulent Rhizoctonia spp. isolates.

Certain hypovirulent Rhizoctonia isolates effectively protect plants against well-known important pathogens among Rhizoctonia isolates as well as against other pathogens. The modes of action involved in this protection include resistance induced in plants by colonization with hypovirulent Rhizoctonia isolates. The qualifications of hypovirulent isolates (efficient protection, rapid growth, effective colonization of the plants, and easy application in the field) provide a significant potential for the development of a commercial microbial preparation for application as biological control agents. Understanding of the modes of action involved in protection is important for improving the various aspects of development and application of such preparations. The hypothesis of the present study is that resistance pathways such as systemic acquired resistance (SAR), induced systemic resistance (ISR), and phytoalexins are induced in plants colonized by the protective hypovirulent Rhizoctonia isolates and are involved in the protection of these plants against pathogenic Rhizoctonia. Changes in protection levels of Arabidopsis thaliana mutants defective in defense-related genes (npr1-1, npr1-2, ndr1-1, npr1-2/ndr1-1, cim6, wrky70.1, snc1, and pbs3-1) and colonized with the hypovirulent Rhizoctonia isolates compared with that of the wild type (wt) plants colonized with the same isolates confirmed the involvement of induced resistance in the protection of the plants against pathogenic Rhizoctonia spp., although protection levels of mutants constantly expressing SAR genes (snc1 and cim6) were lower than that of wt plants. Plant colonization by hypovirulent Rhizoctonia isolates induced elevated expression levels of the following genes: PR5 (SAR), PDF1.2, LOX2, LOX1, CORI3 (ISR), and PAD3 (phytoalexin production), which indicated that all of these pathways were induced in the hypovirulent-colonized plants. When SAR or ISR were induced separately in plants after application of the chemical inducers Bion and methyl jasmonate, respectively, only ISR activation resulted in a higher protection level against the pathogen, although the protection was minor. In conclusion, plant colonization with the protective hypovirulent Rhizoctonia isolates significantly induced genes involved in the SAR, ISR, and phytoalexin production pathways. In the studied system, SAR probably did not play a major role in the mode of protection against pathogenic Rhizoctonia spp.; however, it may play a more significant role in protection against other pathogens.

A novel cost of R gene resistance in the presence of disease.

Resistance responses can impose fitness costs when pests are absent. Here, we test whether the induction of resistance can decrease fitness even in plants under attack; we call this potential outcome a net cost with attack. Using lines in which genetic background was controlled, we investigated whether susceptible Arabidopsis thaliana plants can outperform R gene resistant plants when infected with pathogens. For the R gene RPS2, there was a fitness benefit of resistance in the presence of intraspecific competition, but there was a net cost in the absence of competition: resistant plants produced less seed than susceptible plants even though infected with Pseudomonas syringae. This net cost was primarily due to overcompensation by susceptible plants, which occurred because of a developmental response to infection. For the R gene RPP5, there was no fitness effect of resistance without competition but a net cost when plants were infected with Peronospora parasitica in the presence of competition. This net cost was due to a reduction in the fitness of infected, resistant plants and complete compensation in susceptible plants. A spatially variable model suggests that a trade-off between net benefits and net costs with attack may help explain the persistence of individuals lacking R gene resistance to disease.

Resistance gene analogues of Arabidopsis thaliana: recognition by structure.

Following completion of Arabidopsis thaliana sequencing projects, multiple resistance gene analogues (RGAs) have been identified. In this work a review of the current state of knowledge available in protein databases and scientific articles is presented. Putative resistance genes were identified by using BLAST searches as well as HMM fingerprints (the latter to infer existence of characteristic domains). The representation of all five classes of putative resistance genes in Col-0 ecotype was examined, along with the statistics on RGAs present on all five chromosomes of Arabidopsis thaliana.