Harder, better, faster, stronger: iron strengthens pathogenic bacteria too!

Popeye was right about eating a lot of spinach! Iron, not only present in spinach but also in all other plants, is an essential component of all life forms on earth. The strictly regulated systems in plants that control iron homeostasis and acquisition are different from those in pathogenic bacteria. In Arabidopsis thaliana, multiple basic helix-loop-helix (bHLH) transcription factors, to which bHLH115 and ILR3 (bHLH105) belong, regulate downstream iron regulator target genes (such as FRO2 and IRT1) that mediate iron acquisition under iron-limiting conditions. Contrastingly, when sufficient iron is present, the nucleus-localized iron sensor BRUTUS (BTS) acts together with POPEYE (PYE) or PYE-like proteins to negatively regulate these bHLHs (Selote et al., 2015).

In contrast to the iron acquisition system in plants, pathogenic bacteria secrete iron-chelating siderophores that sequester iron from their hosts. However, the iron acquisition strategy of Pseudomonas syringae pv. tomato (Pst) DC3000 remains to be elucidated. To unravel how P. syringae acquires iron from its host, Xing et al. (2021) focused on the pathogenic effector AvrRps4 initially described by Sohn et al. (2009). To promote infection, P. syringae secretes effector proteins, such as AvrRps4 that is recognized by the immunity receptor pair RESISTANCE TO RALSTONA SOLANACEARUM 1–RESISTANCE TO PSEUDOMONAS SYRINGAE 4. This recognition leads to effector-triggered immunity (ETI) mediated through the ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) family of proteins (Bhandari et al., 2019).

The authors first performed an RNA-sequencing analysis on Arabidopsis Col-0 and eds1 mutant lines infected with the avirulent strain Pst (avrRps4) and demonstrated an EDS1-independent early response in iron homeostasis. Next, using iron-deficiency response mutants, different central iron homeostasis regulators were tested for their role in Pst disease resistance. Notably, ilr3-2 and bts-2 mutants showed, respectively, enhanced resistance and susceptibility to both the virulent Pst and avirulent Pst (avrRps4) strains. These phenotypes were accompanied with decreased and increased apoplastic iron accumulation for ilr3-2 and bts-2 mutants compared with Col-0, respectively.

To further investigate whether AvrRps4 can manipulate iron homeostasis and thereby facilitate bacterial proliferation, the authors used different pathogenic effectors recognized by different immunity receptors and showed that the involvement of BTS was specific for AvrRps4-mediated ETI. Multiple convincing in vitro and in vivo protein–protein interaction and co-expression assays subsequently confirmed that the AvrRps4 C terminus directly binds to BTS, and thereby inhibits BTS-dependent degradation of bHLH115 and ILR3. Moreover, by measuring the iron content after Pst and Pst (avrRps4) infection in diverse ETI-related mutants and transgenic lines over-expressing the effector, the authors showed a clear correlation between increased apoplastic iron content and AvrRps4 Pst infection. Expression studies further indicated that AvrRps4-BTS2-triggered iron accumulation only occurs in the absence of EDS1, whereas BTS is involved in accumulating EDS1 proteins and triggering the immunity response.

Taken together, this study of Xing et al. (2021) provides convincing evidence of the involvement a bacterial pathogenic effector in regulating iron homeostasis by directly targeting a key iron sensor, and thereby strengthening pathogens (Figure).

Figure.

AvrRPS4 binds to BTS2 and accumulates iron and initiates immunity in an EDS1-dependent manner. (Top, left) AvrRps4 interacts with BTS in vivo by co-immunoprecipitation in N. benthamiana leaves. 35S:GFP was used as a negative control. (Top, right) AvrRps4 facilitates apoplastic iron accumulation in the eds1-2 mutant. (Bottom) The bacterial pathogenic effector AvrRps4 targets BTS, leading to impaired BTS fine-tuning of the iron-responsive target genes by which plants accumulate iron and thereby facilitating bacterial growth. In the presence of RPS4/EDS1, BTS is guarded and AvrRPS4 triggers RPS4/EDS1-mediated immunity, limiting iron accumulation and bacterial proliferation (Adapted from Xing et al., [2021], Figures 2, 4 and 6).