HSP90 Contributes to chs3-2D-Mediated Autoimmunity.

Plants employ multi-layered immune system to fight against pathogen infections. Different receptors are able to detect the invasion activities of pathogens, transduce signals to downstream components, and activate defense responses. Among those receptors, nucleotide-binding domain leucine-rich repeat containing proteins (NLRs) are the major intracellular ones. CHILLING SENSITIVE 3 (CHS3) is an Arabidopsis NLR with an additional Lin-11, Isl-1 and Mec-3 (LIM) domain at its C terminus. The gain-of-function mutant, chs3-2D, exhibiting severe dwarfism and constitutively activated defense responses, was selected as a genetic background in this study for a forward genetic screen. A mutant allele of hsp90.2 was isolated as a partial suppressor of chs3-2D, suggesting that HSP90 is required for CHS3-mediated defense signaling. In addition, HSP90 is also required for the autoimmunity of the Dominant Negative (DN)-SNIPER1 and gain-of-function ADR1-L2 D484V transgenic lines, suggesting a broad role for HSP90 in NLR-mediated defense. Overall, our work indicates a larger contribution of HSP90 not only at the sensor, but also the helper NLR levels.

SUSA2 is an F-box protein required for autoimmunity mediated by paired NLRs SOC3-CHS1 and SOC3-TN2.

Both higher plants and mammals rely on nucleotide-binding leucine-rich repeat (NLR) immune receptors to detect pathogens and initiate immunity. Upon effector recognition, plant NLRs oligomerize for defense activation, the mechanism of which is poorly understood. We previously showed that disruption of the E3 ligase, Senescence-Associated E3 Ubiquitin Ligase 1 (SAUL1) leads to the activation of the NLR SOC3. Here, we report the identification of suppressor of saul1 2 (susa2) and susa3 from the saul1-1 suppressor screen. Pairwise interaction analysis suggests that both SUSA proteins interact with components of an SCFSUSA2 E3 ligase complex as well as CHS1 or TN2, truncated NLRs that pair with SOC3. susa2-2 only suppresses the autoimmunity mediated by either CHS1 or TN2, suggesting its specific involvement in SOC3-mediated immunity. In summary, our study indicates links between plant NLRs and an SCF complex that may enable ubiquitination and degradation of unknown downstream components to activate defense.

Assessing Niche Shifts and Conservatism by Comparing the Native and Post-Invasion Niches of Major Forest Invasive Species.

Invasive species experience biotic and abiotic conditions that may (or may not) resemble their native environment. We explored the methodology of determining climatic niches and compared the native and post-invasion niches of four invasive forest pests to determine if these species experienced shifts or changes in their new climatic niches. We used environmental principle components analysis (PCA-env) method to quantify climatic niche shifts, expansions, and temporal changes. Furthermore, we assessed the effect of variable selection in the delineation and comparison of niche space. We found that variable selection influenced the delineation and overlap of each niche, whereas the subset of climatic variables selected from the first two PCA-env axes explained more variance in environmental conditions than the complete set of climatic variables for all four species. Most focal species showed climatic niche shifts in their invasive range and had not yet fully occupied the available niche within the invaded range. Our species varied the proportion of niche overlap between the native and invasive ranges. By comparing native and invasive niches, we can help predict a species' potential range expansion and invasion potential. Our results can guide monitoring and help inform management of these and other invasive species.

A Rapeseed WRKY Transcription Factor Phosphorylated by CPK Modulates Cell Death and Leaf Senescence by Regulating the Expression of ROS and SA-Synthesis-Related Genes.

Salicylic acid (SA) and reactive oxygen species (ROS) are two well-defined inducers of leaf senescence. Here, we identified a novel WRKY transcription factor gene WSR1 (WRKY regulating SA and ROS 1) in Brassica napus (rapeseed) in promoting SA and ROS production, which eventually led to leaf senescence thereafter. Its expression increased in senescing leaves. Ca2+-dependent protein kinase (CPK) 5 and -6 interacted with and phosphorylated BnaWSR1. Overexpression of phosphomimic BnaWSR1 (BnaWSR1ca) in rapeseed protoplasts elicited ROS production and cell death while its ectopic expression in Arabidopsis enhanced SA and ROS levels and, hence, accelerated leaf senescence. Furthermore, BnaWSR1ca activated the expression of Isochorismate Synthase 1 (ICS1), Respiratory Burst Oxidase Homologue (Rboh) D, and Senescence-Associated Gene 14 (SAG14). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays demonstrated that BnaWSR1ca directly bound to promoter regions of ICS1, RbohD, and SAG14. These data have identified a CPK-WSR1 module that integrates SA and ROS to control cell death and leaf senescence.

Determining Spread Rate of Kudzu Bug (Hemiptera: Plataspidae) and Its Associations With Environmental Factors in a Heterogeneous Landscape.

By the end of 2017, kudzu bug was reported in 652 counties in the United States since it was first observed in Georgia in 2009. Modeling its invasion dynamics is valuable to guide management through early detection and prevention of further invasion. Herein, we initially estimated the spread rate of kudzu bug with county-level invasion records and then determined important spatial factors affecting its spread during years 2010-2016. As kudzu bug infests a large heterogeneous area and shows asymmetric spread, we first utilized spatially constrained clustering (SCC), an unsupervised machine learning method, to divide the infested area into eight spatially contiguous and environmentally homogenous neighborhoods. We then used distance regression and boundary displacement methods to estimate the spread rates in all neighborhoods. Finally, we applied multiple regression to determine spatial factors influencing the spread of kudzu bug. The average spread rate reached 76 km/yr by boundary displacement method; however, the rate varied largely among eight neighborhoods (45-144 km/yr). In the southern region of the infested area, host plant density and wind speed were positively associated with the spread rate, whereas mean annual temperature, precipitation in the fall, and elevation had inverse relationships. In the northern region, January minimum temperature, wind speed, and human population density showed positive relationships. This study increases the knowledge on the spread dynamics of kudzu bug. Our research highlights the utility of SCC to determine natural clustering in a large heterogeneous region for better modeling of local spread patterns and determining important factors affecting the invasions.

TIR-NB-LRR immune receptor SOC3 pairs with truncated TIR-NB protein CHS1 or TN2 to monitor the homeostasis of E3 ligase SAUL1.

Intracellular nucleotide binding (NB) and leucine-rich repeat (NLR) proteins function as immune receptors to recognize effectors from pathogens. They often guard host proteins that are the direct targets of those effectors. Recent findings have revealed that a typical NLR sometimes cooperates with another atypical NLR for effector recognition. Here, by using the CRISPR/Cas9 gene editing method, knockout analysis and biochemical assays, we uncovered differential pairings of typical Toll Interleukin1 receptor (TIR) type NLR (TNL) receptor SOC3 with atypical truncated TIR-NB (TN) proteins CHS1 or TN2 to guard the homeostasis of the E3 ligase SAUL1. Overaccumulation of SAUL1 is monitored by the SOC3-TN2 pair, while SAUL1's disappearance is guarded by the SOC3-CHS1 pair. SOC3 forms a head-to-head genomic arrangement with CHS1 and TN2, indicative of transcriptional co-regulation. Such intricate cooperative interactions can probably enlarge the recognition spectrum and increase the functional flexibility of NLRs, which can partly explain the overwhelming occurrence of NLR gene clustering in higher plants.

Differential regulation of TNL-mediated immune signaling by redundant helper CNLs.

Higher plants utilize nucleotide-binding leucine-rich repeat domain proteins (NLRs) as intracellular immune receptors to recognize pathogen-derived effectors and trigger a robust defense. The Activated Disease Resistance 1 (ADR1) family of coiled-coil NLRs (CNLs) have evolved as helper NLRs that function downstream of many TIR-type sensor NLRs (TNLs). Close homologs of ADR1s form the N REQUIREMENT GENE 1 (NRG1) family in Arabidopsis, the function of which is unclear. Through CRISPR/Cas9 gene editing methods, we discovered that the tandemly repeated NRG1A and NRG1B are functionally redundant and operate downstream of TNLs with differential strengths. Interestingly, ADR1s and NRG1s function in two distinct parallel pathways contributing to TNL-specific immunity. Synergistic effects on basal and TNL-mediated defense were detected among ADR1s and NRG1s. An intact P-loop of NRG1s is not required for mediating signals from sensor TNLs, whereas auto-active NRG1A exhibits autoimmunity. Importantly, NRG1s localize to the cytosol and endomembrane network regardless of the presence of effectors, suggesting a cytosolic activation mechanism. Taken together, different sensor TNLs differentially use two groups of helper NLRs, ADR1s and NRG1s, to transduce downstream defense signals.

Negative regulation of resistance protein-mediated immunity by master transcription factors SARD1 and CBP60g.

Salicylic acid (SA) is an essential defence hormone in plants. Upon pathogen infection, induced biosynthesis of SA is mediated by Isochorismate synthase 1 (ICS1), whose gene transcription is controlled mainly through two redundant transcription factors, SAR Deficient 1 (SARD1) and Calmodulin-binding protein 60-like g (CBP60g). Although these master transcription factors regulate not only positive, but also negative regulators of immunity, how they control signaling events downstream of different immune receptors is unclear. Using autoimmune mutants activating immunity mediated by different receptors we show that, although the sard1 cbp60g double mutant almost fully suppresses the activation of defence mediated by suppressor of npr1-1, constitutive 2 (snc2), it strikingly enhances snc1, which carries a gain-of-function mutation in an intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptor. This negative regulation of immunity is achieved through the transcriptional regulation of negative regulators, such as Nudix hydrolase homolog 6 (NUDT6). Our study highlights the diverse roles, especially the negative ones, in the regulation of plant immunity by the two master immune transcription factors SARD1 and CBP60g.

E3 ligase SAUL1 serves as a positive regulator of PAMP-triggered immunity and its homeostasis is monitored by immune receptor SOC3.

In both plants and animals, intracellular nucleotide-binding leucine-rich repeat proteins (NLRs; or Nod-like receptors) serve as immune receptors to recognize pathogen-derived molecules and mount effective immune responses against microbial infections. Plant NLRs often guard the presence or activity of other host proteins, which are the direct virulence targets of pathogen effectors. These guardees are sometimes immune-promoting components such as those in a mitogen-activated protein kinase cascade. Plant E3 ligases serve many roles in immune regulation, but it is unclear whether they can also be guarded by NLRs. Here, we report on an immune-regulating E3 ligase SAUL1, whose homeostasis is monitored by a Toll interleukin 1 receptor (TIR)-type NLR (TNL), SOC3. SOC3 can associate with SAUL1, and either loss or overexpression of SAUL1 triggers autoimmunity mediated by SOC3. By contrast, SAUL1 functions redundantly with its close homolog PUB43 to promote PAMP-triggered immunity (PTI). Taken together, the E3 ligase SAUL1 serves as a positive regulator of PTI and its homeostasis is monitored by the TNL SOC3.

Identification and characterization of plant-specific NAC gene family in canola (Brassica napus L.) reveal novel members involved in cell death.

NAC transcription factors are plant-specific and play important roles in plant development processes, response to biotic and abiotic cues and hormone signaling. However, to date, little is known about the NAC genes in canola (or oilseed rape, Brassica napus L.). In this study, a total of 60 NAC genes were identified from canola through a systematical analysis and mining of expressed sequence tags. Among these, the cDNA sequences of 41 NAC genes were successfully cloned. The translated protein sequences of canola NAC genes with the NAC genes from representative species were phylogenetically clustered into three major groups and multiple subgroups. The transcriptional activities of these BnaNAC proteins were assayed in yeast. In addition, by quantitative real-time RT-PCR, we further observed that some of these BnaNACs were regulated by different hormone stimuli or abiotic stresses. Interestingly, we successfully identified two novel BnaNACs, BnaNAC19 and BnaNAC82, which could elicit hypersensitive response-like cell death when expressed in Nicotiana benthamiana leaves, which was mediated by accumulation of reactive oxygen species. Overall, our work has laid a solid foundation for further characterization of this important NAC gene family in canola.

Identification and functional analysis of mitogen-activated protein kinase kinase kinase (MAPKKK) genes in canola (Brassica napus L.).

Mitogen-activated protein kinase (MAPK) signalling cascades, consisting of three types of reversibly phosphorylated kinases (MAPKKK, MAPKK, and MAPK), are involved in important processes including plant immunity and hormone responses. The MAPKKKs comprise the largest family in the MAPK cascades, yet only a few of these genes have been associated with physiological functions, even in the model plant Arabidopsis thaliana. Canola (Brassica napus L.) is one of the most important oilseed crops in China and worldwide. To explore MAPKKK functions in biotic and abiotic stress responses in canola, 66 MAPKKK genes were identified and 28 of them were cloned. Phylogenetic analysis of these canola MAPKKKs with homologous genes from representative species classified them into three groups (A-C), comprising four MAPKKKs, seven ZIKs, and 17 Raf genes. A further 15 interaction pairs between these MAPKKKs and the downstream BnaMKKs were identified through a yeast two-hybrid assay. The interactions were further validated through bimolecular fluorescence complementation (BiFC) analysis. In addition, by quantitative real-time reverse transcription-PCR, it was further observed that some of these BnaMAPKKK genes were regulated by different hormone stimuli, abiotic stresses, or fungal pathogen treatments. Interestingly, two novel BnaMAPKKK genes, BnaMAPKKK18 and BnaMAPKKK19, which could elicit hypersensitive response (HR)-like cell death when transiently expressed in Nicotiana benthamiana leaves, were successfully identified. Moreover, it was found that BnaMAPKKK19 probably mediated cell death through BnaMKK9. Overall, the present work has laid the foundation for further characterization of this important MAPKKK gene family in canola.

Identification, expression and interaction analyses of calcium-dependent protein kinase (CPK) genes in canola (Brassica napus L.).

BACKGROUND: Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a well-known ubiquitous intracellular secondary messenger in plants. Calcium-dependent protein kinases (CPKs) are Ser/Thr protein kinases found only in plants and some protozoans. CPKs are Ca2+ sensors that have both Ca2+ sensing function and kinase activity within a single protein and play crucial roles in plant development and responses to various environmental stresses. RESULTS: In this study, we mined the available expressed sequence tags (ESTs) of B. napus and identified a total of 25 CPK genes, among which cDNA sequences of 23 genes were successfully cloned from a double haploid cultivar of canola. Phylogenetic analysis demonstrated that they could be clustered into four subgroups. The subcellular localization of five selected BnaCPKs was determined using green fluorescence protein (GFP) as the reporter. Furthermore, the expression levels of 21 BnaCPK genes in response to salt, drought, cold, heat, abscisic acid (ABA), low potassium (LK) and oxidative stress were studied by quantitative RT-PCR and were found to respond to multiple stimuli, suggesting that canola CPKs may be convergence points of different signaling pathways. We also identified and cloned five and eight Clade A basic leucine zipper (bZIP) and protein phosphatase type 2C (PP2C) genes from canola and, using yeast two-hybrid and bimolecular fluorescence complementation (BiFC), determined the interaction between individual BnaCPKs and BnabZIPs or BnaPP2Cs (Clade A). We identified novel, interesting interaction partners for some of the BnaCPK proteins. CONCLUSION: We present the sequences and characterization of CPK gene family members in canola for the first time. This work provides a foundation for further crop improvement and improved understanding of signal transduction in plants.

Identification and characterization of CBL and CIPK gene families in canola (Brassica napus L.).

BACKGROUND: Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a ubiquitous intracellular secondary messenger in plants. Calcineurin B-like proteins (CBLs) are Ca2+ sensors and regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs). Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in canola. RESULTS: In the present study, we identified seven CBL and 23 CIPK genes from canola by database mining and cloning of cDNA sequences of six CBLs and 17 CIPKs. Phylogenetic analysis of CBL and CIPK gene families across a variety of species suggested genome duplication and diversification. The subcellular localization of three BnaCBLs and two BnaCIPKs were determined using green fluorescence protein (GFP) as the reporter. We also demonstrated interactions between six BnaCBLs and 17 BnaCIPKs using yeast two-hybrid assay, and a subset of interactions were further confirmed by bimolecular fluorescence complementation (BiFC). Furthermore, the expression levels of six selected BnaCBL and 12 BnaCIPK genes in response to salt, drought, cold, heat, ABA, methyl viologen (MV) and low potassium were examined by quantitative RT-PCR and these CBL or CIPK genes were found to respond to multiple stimuli, suggesting that the canola CBL-CIPK network may be a point of convergence for several different signaling pathways. We also performed a comparison of interaction patterns and expression profiles of CBL and CIPK in Arabidospsis, canola and rice, to examine the differences between orthologs, highlighting the importance of studying CBL-CIPK in canola as a prerequisite for improvement of this crop. CONCLUSIONS: Our findings indicate that CBL and CIPK family members may form a dynamic complex to respond to different abiotic or hormone signaling. Our comparative analyses of the CBL-CIPK network between canola, Arabidopsis and rice highlight functional differences and the necessity to study CBL-CIPK gene functions in canola. Our data constitute a valuable resource for CBL and CPK genomics.

Identification and analysis of MKK and MPK gene families in canola (Brassica napus L.).

BACKGROUND: Eukaryotic mitogen-activated protein kinase (MAPK/MPK) signaling cascades transduce and amplify environmental signals via three types of reversibly phosphorylated kinases to activate defense gene expression. Canola (oilseed rape, Brassica napus) is a major crop in temperate regions. Identification and characterization of MAPK and MAPK kinases (MAPKK/MKK) of canola will help to elucidate their role in responses to abiotic and biotic stresses. RESULTS: We describe the identification and analysis of seven MKK (BnaMKK) and 12 MPK (BnaMPK) members from canola. Sequence alignments and phylogenetic analyses of the predicted amino acid sequences of BnaMKKs and BnaMPKs classified them into four different groups. We also examined the subcellular localization of four and two members of BnaMKK and BnaMPK gene families, respectively, using green fluorescent protein (GFP) and, found GFP signals in both nuclei and cytoplasm. Furthermore, we identified several interesting interaction pairs through yeast two-hybrid (Y2H) analysis of interactions between BnaMKKs and BnaMPKs, as well as BnaMPK and BnaWRKYs. We defined contiguous signaling modules including BnaMKK9-BnaMPK1/2-BnaWRKY53, BnaMKK2/4/5-BnaMPK3/6-BnaWRKY20/26 and BnaMKK9-BnaMPK5/9/19/20. Of these, several interactions had not been previously described in any species. Selected interactions were validated in vivo by a bimolecular fluorescence complementation (BiFC) assay. Transcriptional responses of a subset of canola MKK and MPK genes to stimuli including fungal pathogens, hormones and abiotic stress treatments were analyzed through real-time RT-PCR and we identified a few of BnaMKKs and BnaMPKs responding to salicylic acid (SA), oxalic acid (OA), Sclerotinia sclerotiorum or other stress conditions. Comparisons of expression patterns of putative orthologs in canola and Arabidopsis showed that transcript expression patterns were generally conserved, with some differences suggestive of sub-functionalization. CONCLUSIONS: We identified seven MKK and 12 MPK genes from canola and examined their phylogenetic relationships, transcript expression patterns, subcellular localization, and protein-protein interactions. Not all expression patterns and interactions were conserved between canola and Arabidopsis, highlighting the limitations of drawing inferences about crops from model species. The data presented here provide the first systematic description of MKK-MPK-WRKY signaling modules in canola and will further improve our understanding of defense responses in general and provide a basis for future crop improvement.