James R. Alfano

Microtubule-associated protein 65-1 (MAP65-1) protein plays an essential role in plant cellular dynamics through impacting stabilization of the cytoskeleton by serving as a crosslinker of microtubules. The role of MAP65-1 in plants has been associated with phenotypic outcomes in response to various environmental stresses. The Arabidopsis MAP65-1 (AtMAP65-1) is a known virulence target of plant bacterial pathogens and is thus a component of plant immunity. Soybean events were generated that carry transgenic alleles for both AtMAP65-1 and GmMAP65-1, the soybean AtMAP65-1 homolog, under control of cauliflower mosaic virus 35S promoter. Both AtMAP65-1 and GmMAP65-1 transgenic soybeans are more resistant to challenges by the soybean bacterial pathogen Pseudomonas syringae pv. glycinea and the oomycete pathogen Phytophthora sojae, but not the soybean cyst nematode, Heterodera glycines. Soybean plants expressing AtMAP65-1 and GmMAP65-1 also display a tolerance to the herbicide oryzalin, which has a mode of action to destabilize microtubules. In addition, GmMAP65-1-expressing soybean plants show reduced cytosol ion leakage under freezing conditions, hinting that ectopic expression of GmMAP65-1 may enhance cold tolerance in soybean. Taken together, overexpression of AtMAP65-1 and GmMAP65-1 confers tolerance of soybean plants to various biotic and abiotic stresses.Copyright (c) 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Root associated microbes are key players in plant health, disease resistance, and nitrogen (N) use efficiency. It remains largely unclear how the interplay of biological and environmental factors affects rhizobiome dynamics in agricultural systems. Here, we quantified the composition of rhizosphere and bulk soil microbial communities associated with maize (Zea mays L.) and soybean (Glycine max L.) in a long-term crop rotation study under conventional fertilization and low N regimes. Over two growing seasons, we evaluated the effects of environmental conditions and several treatment factors on the abundance of rhizosphere and soil colonizing microbial taxa. Time of sampling, host plant species and N fertilization had major effects on microbiomes, while no effect of crop rotation was observed. Using variance partitioning as well as 16S sequence information, we further defined a set of 82 microbial genera and sub-genus groups that show distinct responses to treatment factors. We identified taxa that are highly specific to either maize or soybean rhizospheres, as well as taxa that are sensitive to N fertilization in plant rhizospheres and bulk soil. This study provides insights to harness the full potential of soil microbes in maize and soybean agricultural systems through plant breeding and field management.

Receptor-like proteins (RLPs) and receptor-like kinases (RLKs) are cell surface receptors that are essential for detecting invading pathogens and subsequent activation of plant defence responses. RLPs lack a cytoplasmic kinase domain to trigger downstream signalling leading to host resistance. The RLK SOBIR1 constitutively interacts with the tomato RLP Cf-4, thereby providing Cf-4 with a kinase domain. SOBIR1 is required for Cf-4-mediated resistance to strains of the fungal tomato pathogen Cladosporium fulvum that secrete the effector Avr4. Upon perception of this effector by the Cf-4/SOBIR1 complex, the central regulatory RLK SERK3a is recruited to the complex and defence signalling is triggered. SOBIR1 is also required for RLP-mediated resistance to bacterial, fungal and oomycete pathogens and we hypothesized that SOBIR1 is targeted by effectors of such pathogens to suppress host defence responses. In this study we show that Pseudomonas syringae pv. tomato DC3000 effector AvrPto interacts with Arabidopsis SOBIR1 and its orthologs of tomato and Nicotiana benthamiana, independent of SOBIR1 kinase activity. Interestingly, AvrPto suppresses Arabidopsis SOBIR1-induced cell death in N. benthamiana. Furthermore, AvrPto compromises Avr4-triggered cell death in Cf-4-transgenic N. benthamiana, without affecting Cf-4/SOBIR1/SERK3a complex formation. Our study shows that the RLP co-receptor SOBIR1 is targeted by a bacterial effector, which results in compromised defence responses.

Summary: Pseudomonas syringaetypeIIIeffectors are known to suppress plant immunity to promote bacterial virulence. However, the activities and targets of these effectors are not well understood. We used genetic, molecular, and cell biology methods to characterize the activities, localization, and target of theHopD1 typeIIIeffector inArabidopsis. HopD1 contributes toP. syringaevirulence inArabidopsis and reduces effector‐triggered immunity (ETI) responses but not pathogen‐associated molecular pattern‐triggered immunity (PTI) responses. Plants expressingHopD1 supported increased growth ofETI‐inducingP. syringaestrains compared with wild‐typeArabidopsis. We show thatHopD1 interacts with the membrane‐tetheredArabidopsis transcription factorNTL9 and demonstrate that this interaction occurs at the endoplasmic reticulum (ER). AP. syringae hopD1mutant andETI‐inducingP. syringaestrains exhibited enhanced growth onArabidopsisntl9mutant plants. Conversely, growth ofP. syringaestrains was reduced in plants expressing a constitutively activeNTL9 derivative, indicating thatNTL9 is a positive regulator of plant immunity. Furthermore,HopD1 inhibited the induction ofNTL9‐regulated genes duringETIbut notPTI. HopD1 contributes toP. syringaevirulence in part by targetingNTL9, resulting in the suppression ofETIresponses but notPTIresponses and the promotion of plant pathogenicity.

Summary: The pathogen Pseudomonas syringae requires a type‐III protein secretion system and the effector proteins it injects into plant cells for pathogenesis. The primary role for P. syringae type‐III effectors is the suppression of plant immunity. The P. syringae pv. tomato DC3000 HopK1 type‐III effector was known to suppress the hypersensitive response (HR), a programmed cell death response associated with effector‐triggered immunity. Here we show that DC3000 hopK1 mutants are reduced in their ability to grow in Arabidopsis, and produce reduced disease symptoms. Arabidopsis transgenically expressing HopK1 are reduced in PAMP‐triggered immune responses compared with wild‐type plants. An N‐terminal region of HopK1 shares similarity with the corresponding region in the well‐studied type‐III effector AvrRps4; however, their C‐terminal regions are dissimilar, indicating that they have different effector activities. HopK1 is processed in planta at the same processing site found in AvrRps4. The processed forms of HopK1 and AvrRps4 are chloroplast localized, indicating that the shared N‐terminal regions of these type‐III effectors represent a chloroplast transit peptide. The HopK1 contribution to virulence and the ability of HopK1 and AvrRps4 to suppress immunity required their respective transit peptides, but the AvrRps4‐induced HR did not. Our results suggest that a primary virulence target of these type‐III effectors resides in chloroplasts, and that the recognition of AvrRps4 by the plant immune system occurs elsewhere. Moreover, our results reveal that distinct type‐III effectors use a cleavable transit peptide to localize to chloroplasts, and that targets within this organelle are important for immunity.

Pathogens target important components of host immunity to cause disease. The Pseudomonas syringae type III‐secreted effector HopU1 is a mono‐ADP‐ribosyltransferase required for full virulence on Arabidopsis thaliana . HopU1 targets several RNA‐binding proteins including GRP7, whose role in immunity is still unclear. Here, we show that GRP7 associates with translational components, as well as with the pattern recognition receptors FLS2 and EFR. Moreover, GRP7 binds specifically FLS2 and EFR transcripts in vivo through its RNA recognition motif. HopU1 does not affect the protein–protein associations between GRP7, FLS2 and translational components. Instead, HopU1 blocks the interaction between GRP7 and FLS2 and EFR transcripts in vivo . This inhibition correlates with reduced FLS2 protein levels upon Pseudomonas infection in a HopU1‐dependent manner. Our results reveal a novel virulence strategy used by a microbial effector to interfere with host immunity.

The bacterial pathogenPseudomonas syringaepv. tomato DC3000 must detoxify plant-produced hydrogen peroxide (H₂O₂) in order to survive in its host plant. Candidate enzymes for this detoxification include the monofunctional catalases KatB and KatE and the bifunctional catalase-peroxidase KatG of DC3000. This study shows that KatG is the major housekeeping catalase of DC3000 and provides protection against menadione-generated endogenous H₂O₂. In contrast, KatB rapidly and substantially accumulates in response to exogenous H₂O₂. Furthermore, KatB and KatG have nonredundant roles in detoxifying exogenous H₂O₂and are required for full virulence of DC3000 inArabidopsis thaliana. Therefore, the nonredundant ability of KatB and KatG to detoxify plant-produced H₂O₂is essential for the bacteria to survive in plants. Indeed, a DC3000 catalase triple mutant is severely compromised in its ability to growin planta, and its growth can be partially rescued by the expression ofkatB,katE, orkatG. Interestingly, our data demonstrate that although KatB and KatG are the major catalases involved in the virulence of DC3000, KatE can also provide some protectionin planta. Thus, our results indicate that these catalases are virulence factors for DC3000 and are collectively required for pathogenesis.

Summary: The bacterial plant pathogen Pseudomonas syringae injects effector proteins into plant cells via a type III secretion system (T3SS), which is required for pathogenesis. The protein HrpJ is secreted by P. syringae and is required for a fully functional T3SS. A hrpJ mutant is non‐pathogenic and cannot inject effectors into plant cells or secrete the harpin HrpZ1. Here we show that the hrpJ mutant also cannot secrete the harpins HrpW1 and HopAK1 or the translocator HrpK1, suggesting that these proteins are required in the translocation (injection) of effectors into plant cells. Complementation of the hrpJ mutant with secretion incompetent HrpJ derivatives restores the secretion of HrpZ1 and HrpW1 and the ability to elicit a hypersensitive response, a measure of translocation. However, growth in planta and disease symptom production is only partially restored, suggesting that secreted HrpJ may have a direct role in virulence. Transgenic Arabidopsis plants expressing HrpJ‐HA complemented the virulence phenotype of the hrpJ mutant expressing a secretion incompetent HrpJ derivative and were reduced in their immune responses. Collectively, these data indicate that HrpJ has a dual role in P. syringae: inside bacterial cells HrpJ controls the secretion of translocator proteins and inside plant cells it suppresses plant immunity.