Dissecting the cotranscriptome landscape of plants and their microbiota.

Interactions between plants and neighboring microbial species are fundamental elements that collectively determine the structure and function of the plant microbiota. However, the molecular basis of such interactions is poorly characterized. Here, we colonize Arabidopsis leaves with nine plant-associated bacteria from all major phyla of the plant microbiota and profile cotranscriptomes of plants and bacteria six hours after inoculation. We detect both common and distinct cotranscriptome signatures among plant-commensal pairs. In planta responses of commensals are similar to those of a disarmed pathogen characterized by the suppression of genes involved in general metabolism in contrast to a virulent pathogen. We identify genes that are enriched in the genome of plant-associated bacteria and induced in planta, which may be instrumental for bacterial adaptation to the host environment and niche separation. This study provides insights into how plants discriminate among bacterial strains and lays the foundation for in-depth mechanistic dissection of plant-microbiota interactions.

Author Correction: Multidimensional gene regulatory landscape of a bacterial pathogen in plants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Multidimensional gene regulatory landscape of a bacterial pathogen in plants.

Understanding the gene regulation of plant pathogens is crucial for pest control and thus global food security. An integrated understanding of bacterial gene regulation in the host is dependent on multi-omic datasets, but these are largely lacking. Here, we simultaneously characterized the transcriptome and proteome of a bacterial pathogen in plants. We found a number of bacterial processes affected by plant immunity at the transcriptome and proteome levels. For instance, salicylic acid-mediated plant immunity suppressed the accumulation of proteins comprising the tip component of the bacterial type III secretion system. Interestingly, there were instances of concordant and discordant regulation of bacterial messenger RNAs and proteins. Gene co-expression analysis uncovered previously unknown gene regulatory modules underlying virulence. This study provides molecular insights into the multiple layers of gene regulation that contribute to bacterial growth in planta, and elucidates the role of plant immunity in affecting pathogen responses.

A plant effector-triggered immunity signaling sector is inhibited by pattern-triggered immunity.

Since signaling machineries for two modes of plant-induced immunity, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), extensively overlap, PTI and ETI signaling likely interact. In an Arabidopsis quadruple mutant, in which four major sectors of the signaling network, jasmonate, ethylene, PAD4, and salicylate, are disabled, the hypersensitive response (HR) typical of ETI is abolished when the Pseudomonas syringae effector AvrRpt2 is bacterially delivered but is intact when AvrRpt2 is directly expressed in planta These observations led us to discovery of a network-buffered signaling mechanism that mediates HR signaling and is strongly inhibited by PTI signaling. We named this mechanism the ETI-Mediating and PTI-Inhibited Sector (EMPIS). The signaling kinetics of EMPIS explain apparently different plant genetic requirements for ETI triggered by different effectors without postulating different signaling machineries. The properties of EMPIS suggest that information about efficacy of the early immune response is fed back to the immune signaling network, modulating its activity and limiting the fitness cost of unnecessary immune responses.

Dual regulation of gene expression mediated by extended MAPK activation and salicylic acid contributes to robust innate immunity in Arabidopsis thaliana.

Network robustness is a crucial property of the plant immune signaling network because pathogens are under a strong selection pressure to perturb plant network components to dampen plant immune responses. Nevertheless, modulation of network robustness is an area of network biology that has rarely been explored. While two modes of plant immunity, Effector-Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI), extensively share signaling machinery, the network output is much more robust against perturbations during ETI than PTI, suggesting modulation of network robustness. Here, we report a molecular mechanism underlying the modulation of the network robustness in Arabidopsis thaliana. The salicylic acid (SA) signaling sector regulates a major portion of the plant immune response and is important in immunity against biotrophic and hemibiotrophic pathogens. In Arabidopsis, SA signaling was required for the proper regulation of the vast majority of SA-responsive genes during PTI. However, during ETI, regulation of most SA-responsive genes, including the canonical SA marker gene PR1, could be controlled by SA-independent mechanisms as well as by SA. The activation of the two immune-related MAPKs, MPK3 and MPK6, persisted for several hours during ETI but less than one hour during PTI. Sustained MAPK activation was sufficient to confer SA-independent regulation of most SA-responsive genes. Furthermore, the MPK3 and SA signaling sectors were compensatory to each other for inhibition of bacterial growth as well as for PR1 expression during ETI. These results indicate that the duration of the MAPK activation is a critical determinant for modulation of robustness of the immune signaling network. Our findings with the plant immune signaling network imply that the robustness level of a biological network can be modulated by the activities of network components.

An efficient Agrobacterium-mediated transient transformation of Arabidopsis.

Agrobacterium tumefaciens-mediated transient transformation has been a useful procedure for characterization of proteins and their functions in plants, including analysis of protein-protein interactions. Agrobacterium-mediated transient transformation of Nicotiana benthamiana by leaf infiltration has been widely used due to its ease and high efficiency. However, in Arabidopsis this procedure has been challenging. Previous studies suggested that this difficulty was caused by plant immune responses triggered by perception of Agrobacterium. Here, we report a simple and robust method for Agrobacterium-mediated transient transformation in Arabidopsis. AvrPto is an effector protein from the bacterial plant pathogen Pseudomonas syringae that suppresses plant immunity by interfering with plant immune receptors. We used transgenic Arabidopsis plants that conditionally express AvrPto under the control of a dexamethasone (DEX)-inducible promoter. When the transgenic plants were pretreated with DEX prior to infection with Agrobacterium carrying a ß-glucuronidase (GUS, uidA) gene with an artificial intron and driven by the CaMV 35S promoter, transient GUS expression was dramatically enhanced compared to that in mock-pretreated plants. This transient expression system was successfully applied to analysis of the subcellular localization of a cyan fluorescent protein (CFP) fusion and a protein-protein interaction in Arabidopsis. Our findings enable efficient use of Agrobacterium-mediated transient transformation in Arabidopsis thaliana.

The molecular basis of chromosome orthologies and sex chromosomal differentiation in palaeognathous birds.

Palaeognathous birds (Struthioniformes and Tinamiformes) have morphologically conserved karyotypes and less differentiated ZW sex chromosomes. To delineate interspecific chromosome orthologies in palaeognathous birds we conducted comparative chromosome painting with chicken (Gallus gallus, GGA) chromosome 1-9 and Z chromosome paints (GGA1-9 and GGAZ) for emu, double-wattled cassowary, ostrich, greater rhea, lesser rhea and elegant crested tinamou. All six species showed the same painting patterns: each probe was hybridized to a single pair of chromosomes with the exception that the GGA4 was hybridized to the fourth largest chromosome and a single pair of microchromosomes. The GGAZ was also hybridized to the entire region of the W chromosome, indicating that extensive homology remains between the Z and W chromosomes on the molecular level. Comparative FISH mapping of four Z- and/or W-linked markers, the ACO1/IREBP, ZOV3 and CHD1 genes and the EE0.6 sequence, revealed the presence of a small deletion in the proximal region of the long arm of the W chromosome in greater rhea and lesser rhea. These results suggest that the karyotypes and sex chromosomes of palaeognathous birds are highly conserved not only morphologically, but also at the molecular level; moreover, palaeognathous birds appear to retain the ancestral lineage of avian karyotypes.

Comparison of the Z and W sex chromosomal architectures in elegant crested tinamou (Eudromia elegans) and ostrich (Struthio camelus) and the process of sex chromosome differentiation in palaeognathous birds.

To clarify the process of avian sex chromosome differentiation in palaeognathous birds, we performed molecular and cytogenetic characterization of W chromosome-specific repetitive DNA sequences for elegant crested tinamou (Eudromia elegans, Tinamiformes) and constructed comparative cytogenetic maps of the Z and W chromosomes with nine chicken Z-linked gene homologues for E. elegans and ostrich (Struthio camelus, Struthioniformes). A novel family of W-specific repetitive sequences isolated from E. elegans was found to be composed of guanine- and cytosine-rich 293-bp elements that were tandemly arrayed in the genome as satellite DNA. No nucleotide sequence homologies were found for the Struthioniformes and neognathous birds. The comparative cytogenetic maps of the Z and W chromosomes of E. elegans and S. camelus revealed that there are partial deletions in the proximal regions of the W chromosomes in the two species, and the W chromosome is more differentiated in E. elegans than in S. camelus. These results suggest that a deletion firstly occurred in the proximal region close to the centromere of the acrocentric proto-W chromosome and advanced toward the distal region. In E. elegans, the W-specific repeated sequence elements were amplified site-specifically after deletion of a large part of the W chromosome occurred.