Proteases Underground: Analysis of the Maize Root Apoplast Identifies Organ Specific Papain-Like Cysteine Protease Activity

Abstract

Plant proteases are key regulators of plant cell processes such as seed development, immune responses, senescence and programmed cell death (PCD). Apoplastic papain-like cysteine proteases (PL) are hubs in plant-microbe interactions and play an important role during abiotic stresses. The apoplast is a crucial interface for the interaction between plant and microbes. So far, apoplastic maize PL and their function have been mostly described for aerial parts. In this study, we focused on apoplastic PLCPs in the roots of maize plants. We have analyzed the phylogeny of maize PLCPs and investigated their protein abundance after salicylic acid (SA) treatment. Using activity-based protein profiling (ABPP) we have identified a novel root-specific PLCP belonging to the RD21-like subfamily, as well as three SA activated PLCPs. The root specific PLCP CP1C shares sequence and structural similarities to known CP1-like proteases. Biochemical analysis of recombinant CP1C revealed different substrate specificities and inhibitor affinities compared to the related proteases. This study characterized a root-specific PLCP and identifies differences between the SA-dependent activation of PLCPs in roots and leaves.

Epichloë festucaein mutualistic association withLolium perennesuppresses host apoplastic cysteine protease activity

“…pL1M-F1-LpCP1::2×35S was amplified from LpCP1 (maker-scaffold_2516|ref0039699-exonerate_est2genome-gene-0.3) leaving out the DNA sequence coding for the granulin domain. pL1M-F1-CP1Amut-nogran-mCherry::2×35S is described in Schulze Hüynck et al , 2019. The DNA fragments were ligated as previously described in Weber et al , 2011 and transformed first into E. coli Top10 competent cells and then into A. tumefaciens GV3101 competent cells for overexpression in N. benthamiana .…”

“…Isolation of N. benthamiana apoplastic fluids was performed as described in Schulze Hüynck et al , 2019. In short, three days post Agrobacterium infiltration N. benthamiana leaves were harvested, and vacuum infiltrated with MilliQ water three times for 5 min at 60 mbar with a 2 min interval of atmospheric pressure.…”

“…As a negative control, one set of samples was incubated with an equivalent amount of DMSO instead of DCG-04. The pulldown experiment was subsequently performed as described in Schulze Hüynck et al , 2019.…”

“…The mix was subsequently applied to a flow-through column and the column was washed three times with 12 ml 1x PBS (pH Table S3. The PLCP phylogenetic tree was generated using the identified PLCPs in L. perenne as well as 52 maize PLCP sequences from the MEROPS database (B73 line), six maize PLCPs identified in the early golden bantam line and 39 barley PLCP sequences (Díaz-Mendoza et al, 2014;Rawlings et al, 2018;Schulze Hüynck et al, 2019). One PLCP of each PLCP subfamily from Arabidopsis thaliana were also included in the tree (Richau et al, 2012), as well as four A. thaliana cysteine proteases of the family C13 (legumains) α-VPE (AEC07775.1), β-VPE (OAP12170.1), γ-VPE (OAO96694.1) and δ-VPE (OAP02173.1).…”

The Apoplast: A Key Player in Plant Survival

“…For example, Ziemann et al [185] pointed out that Zip1 peptide, which is released in the presence of PLCP, leads to SA accumulation in maize leaves. Moreover, Schulze Hüynck et al [186] identified a specific PLCP named CP1C in maize root. Surprisingly, CP1C which shares sequence homology to the Arabidopsis RD21 subfamily did not show higher activity after SA treatment of maize roots.…”