A humoral immune response to parasitoid wasps in Drosophila is regulated by JAK/STAT, NF-κB and GATA

Reviewer #1 (Public review):

Summary:

The signaling pathways regulating the immune response to bacteria and fungi have been well characterized in Drosophila. Using the recently identified anti-parasitoid effector Lectin24A as a read-out, this article describes the signaling pathways regulating the humoral response against parasites.

Strengths:

This study reveals a role of JAK-STAT, Toll, and GATA in the fat body in the regulation of Lectin24A. They also observe an enrichment of binding sites for NF-kB, STAT, and GATA factors upstream of ORFs of genes induced upon encapsulation. Based on this observation, they generalize their findings on the involvement of JAK-STAT, Toll, and Gata in the humoral response to encapsulation. Although roles for the Toll and JAK-STAT pathways in capsule formation have previously been identified, the merit of this article is in analyzing the roles of these pathways in the humoral response using a new gene readout that will be a precious tool in the community.

Weaknesses:

The data are mostly convincing, but not always analyzed with sufficient detail; their conclusions should be reinforced by monitoring Lectin24A gene expression by RT-qPCR, by adding additional time points and by using alternative genetic tools. Using read-outs of the Toll and Imd pathways as comparisons is also important. Thus, this paper is interesting and important in advancing our understanding of Drosophila immunity but not yet enough solid to reach definitive conclusions on the proposed claims.

Reviewer #2 (Public review):

Summary:

In a previous study, the investigators had identified through genetic analysis of lines derived from natural populations that lectin-24A was an important gene required for protection against the parasitoid wasp Leptopilina boulardii, albeit only in a specific genetic context depending on an unidentified locus on the third chromosome (Arunkumar, et al., PNAS, 2023). They had documented that the gene is induced upon wasp infection and that the corresponding Lectin-24A binds to the wasp egg prior to hemocyte, mediating a faster encapsulating cellular response. They had identified a polymorphism in susceptible lines that correlated with a 21 nt deficiency in the lectin-24A promoter that removed a proximal NF-kappaB binding site. Here, they follow up this work by first performing a transgenic dissection of this promoter, including the mutations of putative transcription factor binding sites (TFBS) of the JAK-STAT, the Toll pathway, and the GATA family transcription factors. Secondly, they directly affect the expression of genes of the JAK-STAT pathway, of the DIF or Dorsal NF-kappaB transcription factors (and also Relish), and of pannier, the one induced gene of five GATA family members. Of note, the lectin is preferentially expressed in the posterior part of the fat body.

Strengths:

The combination of the analysis of the expression of the lectin-24A gene in cis through mutations in putative TFBS for three families of transcription factors and the analysis in trans of either the genetic pathway (JAK-STAT) or the STAT/DIF/Dorsal/Pannier transcription factors provides a fine-grained description of the regulation of the expression of a humoral effector gene that is induced by parasitoid wasp infestation. Thus, this work goes much beyond the bioinformatics analysis by using a rather thorough experimental approach. The finding of an induction of lectin-24A in the posterior rather than the anterior fat body is interesting yet puzzling. Is it known whether this species of parasitoid wasps deposits its eggs preferentially in the posterior part of the larva?

Weaknesses:

There are some discrepancies between the "cis" and "trans" approaches as regards their effects on basal or induced expression of lectin-24A:

JAK-STAT:
Figure 4D shows that mutating three of six predicted STAT TFBS in the 314 bp promoter leads to a reduction of both basal and induced lectin-24A expression levels, with the gene still being inducible. In contrast, knocking down or out the Drosophila JAK and STAT genes abolished the inducibility of the lectin-24A reporter down or close to basal levels. Conversely, the overactivation of the JAK-STAT pathway led to basal levels that increased to those of induced ones.

Toll pathway:
Figure 4D shows that mutating the proximal Dif-Dorsal TFBS reduces both basal and induced levels of the reporter gene to a common level that is below that of the wild-type basal activity. These data suggest that NF-kappaB signaling is required for both basal and induced expression of Lectin-24A. Affecting either Dif or dorsal gene expression led to opposite changes essentially in the basal expression level of the lectin-24A reporter. Conversely, dorsal overexpression in the fat body and other tissues (hemocytes) led to an enhanced basal expression of the lectin gene.

GATA:
The mutation of the single GATA TFBS in the promoter led to a reduced expression phenotype very similar to that of JAK-STAT TFBS mutations. In contrast, ubiquitous somatic KO mutations of pannier did not affect the basal or induced lectin-24A expression levels. The overactivation of pannier using an allele that cannot be negatively regulated leads to a higher induction of Lectin-24A gene expression, strikingly with basal expression going up to induced levels.

Reviewer #3 (Public review):

Summary:

In this very thorough manuscript, the authors provide further evidence that the lectin-24A gene in Drosophila melanogaster is directly involved in the anti-parasitoid wasp humoral immune reaction.

Strengths:

In this study in particular they use a fluorescent reporter and promoter-bashing to determine how this gene is regulated. They find that JAK/STAT, Pannier, and NF-κB signaling are integral to the regulation of lectin-24A and to the humoral anti-parasitoid immune response. These claims are well supported by the experimental design, results, and analysis.

Weaknesses:

A bit of clarity is needed regarding Figure 4a as well as on the rationale for the lengths of the promoter intervals used.