A Perspective on CRN Proteins in the Genomics Age: Evolution, Classification, Delivery and Function Revisited

Abstract

Plant associated microbes rely on secreted virulence factors (effectors) to modulate host immunity and ensure progressive infection. Amongst the secreted protein repertoires defined and studied in pathogens to date, the CRNs (for CRinkling and Necrosis) have emerged as one of only a few highly conserved protein families, spread across several kingdoms. CRN proteins were first identified in plant pathogenic oomycetes where they were found to be modular factors that are secreted and translocated inside host cells by means of a conserved N-terminal domain. Subsequent localization and functional studies have led to the view that CRN C-termini execute their presumed effector function in the host nucleus, targeting processes required for immunity. These findings have led to great interest in this large protein family and driven the identification of additional CRN-like proteins in other organisms. The identification of CRN proteins and subsequent functional studies have markedly increased the number of candidate CRN protein sequences, expanded the range of phenotypes tentatively associated with function and revealed some of their molecular functions toward virulence. The increased number of characterized CRNs also has presented a set of challenges that may impede significant progress in the future. Here, we summarize our current understanding of the CRNs and re-assess some basic assumptions regarding this protein family. We will discuss the latest findings on CRN biology and highlight exciting new hypotheses that have emanated from the field. Finally, we will discuss new approaches to study CRN functions that would lead to a better understanding of CRN effector biology as well as the processes that lead to host susceptibility and immunity.

Keywords: CR-toxins; CRN; Phytophthora; effectors; immunity; nucleus; oomycetes.

FIGURE 1

CR(N) structure analysis. CRN effectors are modular proteins with an N-terminus thought to be responsible for CRN secretion and translocation into the host and a C-terminus responsible for CRN virulence function(s). (A) CRN N-termini were thought to contain a conserved structure featuring: a signal peptide for secretion; an LXLFLAK domain containing the respective LXLFLAK motif connected with translocation; and a DWL domain that ends in a conserved HVLVVVP motif that marks the end of CRN N-terminus and is thought to be a hot spot for recombination events. In contrast, CRN C-termini were shown to exhibit a large variety of domain structures (not depicted here). (B) Zhang et al. (2016) redefined CRN structure. CRN N-termini (renamed header domains) from the two Phytophthora species analyzed (P. infestans and P. sojae) all feature an Ubiquitin like (Ubl) domain that is thought to be responsible for secretion and translocation into the host cell. CRN C-termini (also named CR-toxin domains) feature distinct domain architectures, having enzymatic origins. The majority of Phytophthora CRN C-termini contained the depicted domain structure (NTPase + HTH + REase). (C) Summary of domain architectures predicted to occur in Phytophthora (from Zhang et al., 2016). The number of CRN proteins with each given domain architecture/composition are indicated between brackets.

FIGURE 2

Schematic representation of current knowns and unknowns in CRN effector biology. CRNs (depicted as colored circles) are highly expressed and regulated during infection, suggesting transcriptional control. It has been suggested that CRN could be regulated via siRNAs and PTMs, namely phosphorylation (green stars). A wide variety of CRNs have been identified as being phosphorylated in pathogen structures. However, the post-translational status inside plant cells or the apoplast (during transit) remains unknown. CRN secretion and translocation mechanisms remain widely uncharacterized. CRN N-termini were shown to be sufficient to mediate protein secretion and translocation into plant cells. The presence of the LXLFLAK motif was also shown to be required for this process. However, if CRN translocation is achieved in haustoria or if CRN predicted signal peptides are functional remains unclear. CRNs target host nuclear processes, but the mechanisms of trafficking into the nucleus, remain unknown. Importins mediate nuclear import by binding Nuclear Localization signals (NLSs), present in most proteins destined for the nucleus. However, import of CRNs without predicted NLSs has been observed. CRNs have been shown to mediate or suppress cell death processes. Besides proteinaceous nuclear host targets, CRNs have also been shown to target host DNA. Diverse CRNs were shown to form complexes in plant tissues. However, the nature of these dimers with regards to exact composition remains unclear.