One Sentence SummaryWe demonstrate that continued presence of CXCR5 +/+ TFH cells throughout chronic viral infection is dispensable for maintenance of overall virus-specific antibody titers but is vital for the generation of virus-neutralizing antibodies and eventual control of the infection.

Highlights d Chronic infection reveals sustained clonal diversity of induced antibodies d Antibody repertoires attain a personalized signature during chronic infection d Chronic viral infection shows sustained GC response and PC differentiation d Chronic infection selects for higher-affinity antibodies than acute infection

Bradyrhizobium diazoefficiens USDA 110 (formerly named Bradyrhizobium japonicum) can fix dinitrogen when living as an endosymbiont in root nodules of soybean and some other legumes. Formation of a functional symbiosis relies on a defined developmental program mediated by controlled gene expression in both symbiotic partners. In contrast to other well-studied Rhizobium-legume model systems that have been thoroughly examined by means of genetically tagged strains, analysis of B. diazoefficiens host infection has been impaired due to the lack of suitable tagging systems. Here, we describe the construction of B. diazoefficiens strains constitutively expressing single-copy genes for fluorescent proteins (eBFP2, mTurquoise2, GFP+, sYFP2, mCherry, HcRed) and enzymes (GusA, LacZ). For stable inheritance, the constructs were recombined into the chromosome. Effectiveness and versatility of the tagged strains was demonstrated in plant infection assays. (i) The infection process was followed from root-hair attachment to colonization of nodule cells with epifluorescent microscopy. (ii) Monitoring mixed infections with two strains producing different fluorescent proteins allowed rapid analysis of nodule occupancy and revealed that the majority of nodules contained clonal populations. (iii) Microscopic analysis of nodules induced by fluorescent strains provided evidence for host-dependent control of B. diazoefficiens bacteroid morphology in nodules of Aeschynomene afraspera and Arachis hypogaea (peanut), as deduced from their altered morphology compared with bacteroids in soybean nodules.

Phylogenetically diverse bacteria respond to various stress conditions by mounting a general stress response (GSR) resulting in the induction of protection or damage repair functions. In α-proteobacteria, the GSR is induced by a regulatory cascade consisting of the extracytoplasmic function (ECF) σ factor σ, its anti-σ factor NepR, and the anti-anti-σ factor PhyR. We have reported previously that σ and PhyR of Bradyrhizobium diazoefficiens (formerly named Bradyrhizobium japonicum), the nitrogen-fixing root nodule symbiont of soybean and related legumes, are required for efficient symbiosis; however, the precise role of the GSR remained undefined. Here, we analyze the symbiotic defects of a B. diazoefficiens mutant lacking σ by comparing distinct infection stages of enzymatically or fluorescently tagged wild-type and mutant bacteria. Although root colonization and root hair curling were indistinguishable, the mutant was not competitive, and showed delayed development of emerging nodules and only a few infection threads. Consequently, many of the mutant-induced nodules were aborted, empty, or partially colonized. Congruent with these results, we found that σ was active in bacteria present in root-hair-entrapped microcolonies and infection threads but not in root-associated bacteria and nitrogen-fixing bacteroids. We conclude that GSR-controlled functions are crucial for synchronization of infection thread formation, colonization, and nodule development.

Persistent virus infections with non‐ or poorly cytopathic viruses are commonly associated with B cell dysregulations. These include the induction of hypergammaglobulinemia and the emergence of virus‐unspecific antibodies. These seemingly unspecific antibody responses interfere with the virus‐specific humoral immunity and contribute to delayed virus control. Whether these virus‐unspecific antibodies are induced in the B cell follicle or at extrafollicular sites and whether one specific CD4 T cell subset is involved in the polyclonal B cell activation is unclear. Here we studied virus‐unrelated IgG antibody responses against self or foreign antigens in the context of persistent lymphocytic choriomeningitis virus (LCMV) infection. We found that the LCMV‐unspecific antibody response is short‐lived and induced predominantly at extrafollicular sites and depends on the presence of LCMV‐specific CD4 T cells. Our data support a scenario in which activated, virus‐specific CD4 T cells provide help to non‐specific B cells at extrafollicular sites, supporting the production of virus unspecific IgG antibodies during persistent viral infection.