Pseudomonas syringae pv. tomato DC3000 Type III Secretion Effector Polymutants Reveal an Interplay between HopAD1 and AvrPtoB.

The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered plant innate immune system by injecting a complex repertoire of type III secretion effector (T3E) proteins. Beyond redundancy and interplay, individual T3Es may interact with multiple immunity-associated proteins, rendering their analysis challenging. We constructed a Pst DC3000 polymutant lacking all 36 T3Es and restored individual T3Es or their mutants to explore the interplay among T3Es. The weakly expressed T3E HopAD1 was sufficient to elicit immunity-associated cell death in Nicotiana benthamiana. HopAD1-induced cell death was suppressed partially by native AvrPtoB and completely by AvrPtoBM3, which has mutations disrupting its E3 ubiquitin ligase domain and two known domains for interacting with immunity-associated kinases. AvrPtoBM3 also gained the ability to interact with the immunity-kinase MKK2, which is required for HopAD1-dependent cell death. Thus, AvrPtoB has alternative, competing mechanisms for suppressing effector-triggered plant immunity. This approach allows the deconvolution of individual T3E activities.

Pto kinase binds two domains of AvrPtoB and its proximity to the effector E3 ligase determines if it evades degradation and activates plant immunity.

The tomato--Pseudomonas syringae pv. tomato (Pst)--pathosystem is one of the best understood models for plant-pathogen interactions. Certain wild relatives of tomato express two closely related members of the same kinase family, Pto and Fen, which recognize the Pst virulence protein AvrPtoB and activate effector-triggered immunity (ETI). AvrPtoB, however, contains an E3 ubiquitin ligase domain in its carboxyl terminus which causes degradation of Fen and undermines its ability to activate ETI. In contrast, Pto evades AvrPtoB-mediated degradation and triggers ETI in response to the effector. It has been reported recently that Pto has higher kinase activity than Fen and that this difference allows Pto to inactivate the E3 ligase through phosphorylation of threonine-450 (T450) in AvrPtoB. Here we show that, in contrast to Fen which can only interact with a single domain proximal to the E3 ligase of AvrPtoB, Pto binds two distinct domains of the effector, the same site as Fen and another N-terminal domain. In the absence of E3 ligase activity Pto binds to either domain of AvrPtoB to activate ETI. However, the presence of an active E3 ligase domain causes ubiquitination of Pto that interacts with the domain proximal to the E3 ligase, identical to ubiquitination of Fen. Only when Pto binds its unique distal domain can it resist AvrPtoB-mediated degradation and activate ETI. We show that phosphorylation of T450 is not required for Pto-mediated resistance in vivo and that a kinase-inactive version of Pto is still capable of activating ETI in response to AvrPtoB. Our results demonstrate that the ability of Pto to interact with a second site distal to the E3 ligase domain in AvrPtoB, and not a higher kinase activity or T450 phosphorylation, allows Pto to evade ubiquitination and to confer immunity to Pst.

Temperature-dependent regulation of flowering by antagonistic FLM variants.

The appropriate timing of flowering is crucial for plant reproductive success. It is therefore not surprising that intricate genetic networks have evolved to perceive and integrate both endogenous and environmental signals, such as carbohydrate and hormonal status, photoperiod and temperature. In contrast to our detailed understanding of the vernalization pathway, little is known about how flowering time is controlled in response to changes in the ambient growth temperature. In Arabidopsis thaliana, the MADS-box transcription factor genes FLOWERING LOCUS M (FLM) and SHORT VEGETATIVE PHASE (SVP) have key roles in this process. FLM is subject to temperature-dependent alternative splicing. Here we report that the two main FLM protein splice variants, FLM-ß and FLM-δ, compete for interaction with the floral repressor SVP. The SVP-FLM-ß complex is predominately formed at low temperatures and prevents precocious flowering. By contrast, the competing SVP-FLM-δ complex is impaired in DNA binding and acts as a dominant-negative activator of flowering at higher temperatures. Our results show a new mechanism that controls the timing of the floral transition in response to changes in ambient temperature. A better understanding of how temperature controls the molecular mechanisms of flowering will be important to cope with current changes in global climate.

Nonhost resistance of tomato to the bean pathogen Pseudomonas syringae pv. syringae B728a is due to a defective E3 ubiquitin ligase domain in avrptobb728a.

The bean pathogen Pseudomonas syringae pv. syringae B728a expresses homologs of the type III effectors AvrPto and AvrPtoB, either of which can trigger resistance in tomato cultivars expressing Pto and Prf genes. We found that strain B728a also elicits nonhost resistance in tomato cultivars VFNT Cherry and Moneymaker that lack Pto but express other members of the Pto family (e.g., SlFen and SlPtoC). Here, we show that the AvrPtoB homolog from B728a, termed AvrPtoBB728a (also known as HopAB1), is recognized by 'VFNT Cherry' and 'Moneymaker' when the effector is expressed in P. syringae pv. syringae 61, a strain lacking the avrPto or avrPtoB homolog. Using a gene-silencing approach, this recognition was shown to involve one or more Pto family members and Prf. AvrPtoBB728a interacted with SlFen, SlPtoC, and SlPtoD, in addition to Pto, in a yeast two-hybrid assay. In P. syringae pv. tomato DC3000, the C-terminal domain of AvrPtoB is an E3 ubiquitin ligase that ubiquitinates Fen, causing its degradation and leading to disease susceptibility. Although the C-terminal domain of AvrPtoBB728a shares 69% amino acid identity with that of AvrPtoB, we found that it has greatly reduced E3 ligase activity and is unable to ubiquitinate Fen in an in vitro ubiquitination assay. Thus, the nonhost resistance of 'VFNT Cherry' and 'Moneymaker' to B728a appears to be due to recognition of AvrPtoBB728 as a result of the effector's reduced E3 ligase activity, which prevents it from facilitating degradation of a Pto family member. We speculate that the primary plant host of B728a lacks a Fen-like protein and that, therefore, the E3 ligase of AvrPtoBB728 was unnecessary for pathogenicity and has diverged and become ineffective.

Synteny-based mapping-by-sequencing enabled by targeted enrichment.

Mapping-by-sequencing, as implemented in SHOREmap ('SHOREmapping'), is greatly accelerating the identification of causal mutations. The original SHOREmap approach based on resequencing of bulked segregants required a highly accurate and complete reference sequence. However, current whole-genome or transcriptome assemblies from next-generation sequencing data of non-model organisms do not produce chromosome-length scaffolds. We have therefore developed a method that exploits synteny with a related genome for genetic mapping. We first demonstrate how mapping-by-sequencing can be performed using a reduced number of markers, and how the associated decrease in the number of markers can be compensated for by enrichment of marker sequences. As proof of concept, we apply this method to Arabidopsis thaliana gene models ordered by synteny with the genome sequence of the distant relative Brassica rapa, whose genome has several large-scale rearrangements relative to A. thaliana. Our approach provides an alternative method for high-resolution genetic mapping in species that lack finished genome reference sequences or for which only RNA-seq assemblies are available. Finally, for improved identification of causal mutations by fine-mapping, we introduce a new likelihood ratio test statistic, transforming local allele frequency estimations into a confidence interval similar to conventional mapping intervals.

Structural analysis of Pseudomonas syringae AvrPtoB bound to host BAK1 reveals two similar kinase-interacting domains in a type III Effector.

To infect plants, Pseudomonas syringae pv. tomato delivers ~30 type III effector proteins into host cells, many of which interfere with PAMP-triggered immunity (PTI). One effector, AvrPtoB, suppresses PTI using a central domain to bind host BAK1, a kinase that acts with several pattern recognition receptors to activate defense signaling. A second AvrPtoB domain binds and suppresses the PTI-associated kinase Bti9 but is conversely recognized by the protein kinase Pto to activate effector-triggered immunity. We report the crystal structure of the AvrPtoB-BAK1 complex, which revealed structural similarity between these two AvrPtoB domains, suggesting that they arose by intragenic duplication. The BAK1 kinase domain is structurally similar to Pto, and a conserved region within both BAK1 and Pto interacts with AvrPtoB. BAK1 kinase activity is inhibited by AvrPtoB, and mutations at the interaction interface disrupt AvrPtoB virulence activity. These results shed light on a structural mechanism underlying host-pathogen coevolution.

The genome of Tetranychus urticae reveals herbivorous pest adaptations.

The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and an extreme record of pesticide resistance. Here we present the completely sequenced and annotated spider mite genome, representing the first complete chelicerate genome. At 90 megabases T. urticae has the smallest sequenced arthropod genome. Compared with other arthropods, the spider mite genome shows unique changes in the hormonal environment and organization of the Hox complex, and also reveals evolutionary innovation of silk production. We find strong signatures of polyphagy and detoxification in gene families associated with feeding on different hosts and in new gene families acquired by lateral gene transfer. Deep transcriptome analysis of mites feeding on different plants shows how this pest responds to a changing host environment. The T. urticae genome thus offers new insights into arthropod evolution and plant-herbivore interactions, and provides unique opportunities for developing novel plant protection strategies.

Orchestration of the floral transition and floral development in Arabidopsis by the bifunctional transcription factor APETALA2.

The Arabidopsis thaliana transcription factor APETALA2 (AP2) has numerous functions, including roles in seed development, stem cell maintenance, and specification of floral organ identity. To understand the relationship between these different roles, we mapped direct targets of AP2 on a genome-wide scale in two tissue types. We find that AP2 binds to thousands of loci in the developing flower, many of which exhibit AP2-dependent transcription. Opposing, logical effects are evident in AP2 binding to two microRNA genes that influence AP2 expression, with AP2 positively regulating miR156 and negatively regulating miR172, forming a complex direct feedback loop, which also included all but one of the AP2-like miR172 target clade members. We compare the genome-wide direct target repertoire of AP2 with that of SCHLAFMUTZE, a closely related transcription factor that also represses the transition to flowering. We detect clear similarities and important differences in the direct target repertoires that are also tissue specific. Finally, using an inducible expression system, we demonstrate that AP2 has dual molecular roles. It functions as both a transcriptional activator and repressor, directly inducing the expression of the floral repressor AGAMOUS-LIKE15 and directly repressing the transcription of floral activators like SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1.

Just say no: floral repressors help Arabidopsis bide the time.

Floral repressors ensure correct reproductive timing by safeguarding against premature flowering. In the past decade, several mechanisms of floral repression have come to light. Discrimination between direct and indirect repressors has been facilitated by increasing the use of chromatin immunoprecipitation assays. Certain MADS-domain transcription factors such as SHORT VEGETATIVE PHASE and FLOWERING LOCUS C bind directly to target euchromatin to repress specific loci including FLOWERING LOCUS T (FT) and FD. The AP2-domain transcription factor TEMPRANILLO 1 has also been shown to directly repress FT by binding its 5' UTR. We highlight emerging systems level approaches, including genome-scale direct binding studies (ChIP-chip and ChIP-Seq), which stand out in their promise to elucidate the complex network underlying the transition to flowering at an unprecedented level.

Repression of flowering by the miR172 target SMZ.

A small mobile protein, encoded by the FLOWERING LOCUS T (FT) locus, plays a central role in the control of flowering. FT is regulated positively by CONSTANS (CO), the output of the photoperiod pathway, and negatively by FLC, which integrates the effects of prolonged cold exposure. Here, we reveal the mechanisms of regulation by the microRNA miR172 target SCHLAFMUTZE (SMZ), a potent repressor of flowering. Whole-genome mapping of SMZ binding sites demonstrates not only direct regulation of FT, but also of many other flowering time regulators acting both upstream and downstream of FT, indicating an important role of miR172 and its targets in fine tuning the flowering response. A role for the miR172/SMZ module as a rheostat in flowering time is further supported by SMZ binding to several other genes encoding miR172 targets. Finally, we show that the action of SMZ is completely dependent on another floral repressor, FLM, providing the first direct connection between two important classes of flowering time regulators, AP2- and MADS-domain proteins.

Export of FT protein from phloem companion cells is sufficient for floral induction in Arabidopsis.

Several endogenous and environmental factors need to be integrated to time the onset of flowering. Genetic and molecular analyses, primarily in Arabidopsis thaliana and rice, have shown that CONSTANS (CO) and FLOWERING LOCUS T (FT) play central roles in photoperiod-dependent flowering. The overall picture is that CO acts in the phloem companion cells of leaves and that its main effect is to induce FT mRNA in these cells. Surprisingly, FT, a small globular protein of 20 kDa, interacts at the shoot apex with the bZIP transcription factor FLOWERING LOCUS D (FD) to induce downstream targets. Given that green fluorescent protein (GFP), which as a monomer is 27 kDa, can be easily exported to sink tissue including flowers when expressed in phloem companion cells, the latter finding strongly implied that FT protein is the mobile floral-inductive signal. In agreement with this hypothesis, an FT-GFP fusion, just like GFP, can be exported from the phloem of both rice and Arabidopsis. It has been unknown, however, whether mobile FT protein is sufficient for transmitting the flowering signal. Here we show that FT mRNA is required in phloem companion cells where it acts partially redundant with its paralog TWIN SISTER OF FT (TSF) to induce flowering. Furthermore, we have devised a method that uncouples FT mRNA and protein effects in vivo. We demonstrate that export of FT protein from phloem companion cells is sufficient to induce flowering.

The enteropathogenic Escherichia coli (EPEC) Map effector is imported into the mitochondrial matrix by the TOM/Hsp70 system and alters organelle morphology.

Enteropathogenic Escherichia coli (EPEC) is a human intestinal pathogen and a major cause of diarrhoea, particularly among infants in developing countries. EPEC target the Map and EspF multifunctional effector proteins to host mitochondria - organelles that play crucial roles in regulating cellular processes such as programmed cell death (apoptosis). While both molecules interfere with the organelles ability to maintain a membrane potential, EspF plays the predominant role and is responsible for triggering cell death. To learn more about the Map-mitochondria interaction, we studied Map localization to mitochondria with purified mitochondria (from mammalian and yeast cells) and within intact yeast. This revealed that (i) Map targeting is dependent on the predicted N-terminal mitochondrial targeting sequence, (ii) the N-terminal 44 residues are sufficient to target proteins to mitochondria and (iii) Map import involves the mitochondrial outer membrane translocase (Tom22 and Tom40), the mitochondrial membrane potential, and the matrix chaperone, mtHsp70. These results are consistent with Map import into the mitochondria matrix via the classical import mechanism. As all known, Map-associated phenotypes in mammalian cells are independent of mitochondrial targeting, this may indicate that import serves as a mechanism to remove Map from the cytoplasm thereby regulating cytoplasmic function. Intriguingly, Map, but not EspF, alters mitochondrial morphology with deletion analysis revealing important roles for residues 101-152. Changes in mitochondrial morphology have been linked to alterations in the ability of these organelles to regulate cellular processes providing a possible additional role for Map import into mitochondria.