Recombineering and stable integration of the Pseudomonas syringae pv. syringae 61 hrp/hrc cluster into the genome of the soil bacterium Pseudomonas fluorescens Pf0-1

Many Gram-negative bacteria use a type III secretion system (T3SS) to establish associations with their hosts. The T3SS is a conduit for direct injection of type-III effector proteins into host cells, where they manipulate the host for the benefit of the infecting bacterium. For plant-associated pathogens, the variations in number and amino acid sequences of type-III effectors, as well as their functional redundancy, make studying type-III effectors challenging. To mitigate this challenge, we developed a stable delivery system for individual or defined sets of type-III effectors into plant cells. We used recombineering and Tn5-mediated transposition to clone and stably integrate, respectively, the complete hrp/hrc region from Pseudomonas syringae pv. syringae 61 into the genome of the soil bacterium Pseudomonas fluorescens Pf0-1. We describe our development of Effector-to-Host Analyzer (EtHAn), and demonstrate its utility for studying effectors for their in planta functions.Keywords: Pseudomonas syringae, plant defense, virulence, type-III secretion system, hrp/hrc, type-III effector

Identification of candidate target proteins of type III effectors

Many plant-associated Gram-negative bacteria, such a Pseudomonas syringae pv tomato DC3000 (PtoDC3000), use a type III secretion system (T3SS) to establish a host-microbe relationship with compatible plants. The T3SS resembles a molecular injection mechanism that allows the bacteria to deliver type III effectors (T3Es) directly into the cell of the host plant. These effectors interact with native proteins inside the plant cell in order to dampen the plant’s immunity mechanisms. We are determining the interaction of two T3Es from PtoDC3000 and their candidate target proteins using a yeast two-hybrid assay

Mutualistic Co-evolution of Type III Effector Genes in Sinorhizobium fredii and Bradyrhizobium japonicum

Two diametric paradigms have been proposed to model the molecular co-evolution of microbial mutualists and their eukaryotic hosts. In one, mutualist and host exhibit an antagonistic arms race and each partner evolves rapidly to maximize their own fitness from the interaction at potential expense of the other. In the opposing model, conflicts between mutualist and host are largely resolved and the interaction is characterized by evolutionary stasis. We tested these opposing frameworks in two lineages of mutualistic rhizobia, Sinorhizobium fredii and Bradyrhizobium japonicum. To examine genes demonstrably important for host-interactions we coupled the mining of genome sequences to a comprehensive functional screen for type III effector genes, which are necessary for many Gram-negative pathogens to infect their hosts. We demonstrate that the rhizobial type III effector genes exhibit a surprisingly high degree of conservation in content and sequence that is in contrast to those of a well characterized plant pathogenic species. This type III effector gene conservation is particularly striking in the context of the relatively high genome-wide diversity of rhizobia. The evolution of rhizobial type III effectors is inconsistent with the molecular arms race paradigm. Instead, our results reveal that these loci are relatively static in rhizobial lineages and suggest that fitness conflicts between rhizobia mutualists and their host plants have been largely resolved

HHMI Report 2011 Andres Alvarez.docx

Many plant-associated Gram-negative bacteria, such a Pseudomonas syringae pv tomato DC3000 (PtoDC3000), use a type III secretion system (T3SS) to establish a host-microbe relationship with compatible plants. The T3SS resembles a molecular injection mechanism that allows the bacteria to deliver type III effectors (T3Es) directly into the cell of the host plant. These effectors interact with native proteins inside the plant cell in order to dampen the plant’s immunity mechanisms. We are determining the interaction of two T3Es from PtoDC3000 and their candidate target proteins using a yeast two-hybrid assay

HHMI Report 2011 Andres Alvarez.pdf

Many plant-associated Gram-negative bacteria, such a Pseudomonas syringae pv tomato DC3000 (PtoDC3000), use a type III secretion system (T3SS) to establish a host-microbe relationship with compatible plants. The T3SS resembles a molecular injection mechanism that allows the bacteria to deliver type III effectors (T3Es) directly into the cell of the host plant. These effectors interact with native proteins inside the plant cell in order to dampen the plant’s immunity mechanisms. We are determining the interaction of two T3Es from PtoDC3000 and their candidate target proteins using a yeast two-hybrid assay

Mutualistic Co-evolution of Type III Effector Genes in <em>Sinorhizobium fredii</em> and <em>Bradyrhizobium japonicum</em>

<div><p>Two diametric paradigms have been proposed to model the molecular co-evolution of microbial mutualists and their eukaryotic hosts. In one, mutualist and host exhibit an antagonistic arms race and each partner evolves rapidly to maximize their own fitness from the interaction at potential expense of the other. In the opposing model, conflicts between mutualist and host are largely resolved and the interaction is characterized by evolutionary stasis. We tested these opposing frameworks in two lineages of mutualistic rhizobia, <i>Sinorhizobium fredii</i> and <i>Bradyrhizobium japonicum</i>. To examine genes demonstrably important for host-interactions we coupled the mining of genome sequences to a comprehensive functional screen for type III effector genes, which are necessary for many Gram-negative pathogens to infect their hosts. We demonstrate that the rhizobial type III effector genes exhibit a surprisingly high degree of conservation in content and sequence that is in contrast to those of a well characterized plant pathogenic species. This type III effector gene conservation is particularly striking in the context of the relatively high genome-wide diversity of rhizobia. The evolution of rhizobial type III effectors is inconsistent with the molecular arms race paradigm. Instead, our results reveal that these loci are relatively static in rhizobial lineages and suggest that fitness conflicts between rhizobia mutualists and their host plants have been largely resolved.</p> </div

Statistics for genome mining for T3E-encoding genes.

*<p>A trained Hidden Markov Model (HMM) was used to identify candidate <i>tts</i>-boxes;</p>†<p>CDSs within 10 kb and encoded on the same strand as the predicted <i>tts</i>-box were identified;</p>‡<p>T3E-encoding genes based on T3SS-dependent elicitation of HR by <i>Pt</i>oDC3000 in Arabidopsis Col-0.</p

T3E of <i>S. fredii</i> and <i>B. japonicum</i> have high levels of within-family amino acid identity.

<p>(A) Balloon plots of within-family amino acid conservation for translated T3Es. The percent amino acid identity was calculated for all pairwise comparisons within each family (y-axis) and plotted according to the number of members within families (x-axis). The sizes of the balloons are scaled with the largest representing 162 pairwise comparisons (the smallest balloons were enlarged). Unconfirmed T3E and pseudogene sequences were not included in the comparisons. (B) Kolmogorov–Smirnov test for all pairwise comparisons (connected by lines) of the distributions depicted in panel (A). Boxed <i>p</i>-values are significant (Bonferonni adjusted α level = 0.0083). (C) An F test for linear hypothesis was used to test for differences in percent amino acid identity for translated T3Es and core genes within each group. All <i>p</i>-values are significant (Bonferonni adjusted α level = 0.0125).</p

Distribution of T3E families in rhizobia.

<p>The T3E family names are listed across the top with strains of (A) <i>S. fredii</i> and (B) <i>B. japonicum</i> listed down the side. Boxes are color-coded as indicated in the key; white boxes = no detectable homolog. Conservation of T3Es is also color-coded (bars below each chart) as indicated.</p