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\begin{document}

\title{PREreview of bioRxiv article ``Subfamily-specific functionalization of
diversified immune receptors in wild barley''}



\author[1]{Sophien Kamoun}%
\affil[1]{The Sainsbury Laboratory}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\selectlanguage{english}
\begin{abstract}
This is a review of Maekawa et al. bioRxiv 293050; doi:
\url{https://doi.org/10.1101/352278} posted on June 20, 2018. In this
paper, the authors mined the transcriptomes of 50 different accession of
wild barley, generating a rich library of natural variants of the MLA
immune receptor---a classical nucleotide-binding domain and leucine-rich
repeat-containing (NLR) protein. They grouped the MLA variants in two
subfamilies with all receptors known to be effective against the powdery
mildew fungus grouping in one subfamily.%
\end{abstract}%



\sloppy


\par\null

\section*{Summary}

{\label{216049}}\par\null

In plants, intracellular immune receptors of the NLR
(nucleotide-binding, leucine-rich repeat) family are modular proteins
that monitor translocated pathogen effector proteins and activate immune
responses typified by the hypersensitive cell death. One of these NLR
proteins is MLA, a member of a group of proteins that carry a
coiled-coil (CC) domain prior to the nucleotide-binding (NB) and
leucine-rich repeat (LRR) domains. In this paper, the authors mined the
transcriptome of 50 different accession of wild barley, generating a
rich library of natural variants of the MLA immune receptor. They found
a pattern of diversification in the CC domain, which they argue might be
related to functional diversification of these receptors. Furthermore,
they detected positive selection signals in the LRR region of MLA, which
is thought to confer recognition specificity to the pathogen.

The findings represent an excellent example of molecular evolution in
plant NLRs and the new receptor variants uncovered by this study have
the potential to help in the quest for durable resistance against
different pathogen strains.

\par\null

There are two parts in this study: (1) the generation of a sequence
library of natural variants of the MLA gene and the analyses to identify
signatures of selection and diversification; (2) a combination of
secondary structure prediction and functional analyses of the CC domains
to complement the first part.

\par\null

Our general view is that the first part is interesting and has yielded
exciting molecular evolution findings. Figure S2, for instance, is truly
beautiful with MLA being fairly conserved in its general structure yet
so diverse in terms of amino acid sequences. However, the second part
would benefit from revising the methodology related to the structural
analyses of the CC domains. Also, the functional analyses are limited to
autoactivity assays of the CC-domains. It's not surprising that all of
the assayed domains trigger cell death given that the CC domains of
wheat MLA-like genes Sr33 and Sr50, which group outside the two clades
described here, are also autoactive. Given that all of the assayed
domains are autoactive, and appear to be conserved across the broader
MLA family including wheat homologs, it is difficult to accept several
of the conclusions proposed in this manuscript. For example, the title
(``subfamily-specific functionalization'') might be misleading given
that no pathogen or effector related functional data on subfamily 2 has
been reported.

\section*{\(\)Findings and
comments}

{\label{457509}}\par\null

\subsection*{(1)~~Overall comments}

{\label{408285}}

The lack of functional analyses with pathogens and/or effectors and the
fact that the study does not identify a differential phenotype between
the reported subfamilies, do not support the statements about functional
diversification.

\par\null

CC domain differences are proposed to drive the diversification of the
MLA receptor. However, the phylogeny of the conserved NB domain does not
fully recapitulate this diversification.

\par\null

The manuscript would benefit from a more thorough functional assessment
of a range of CC domains. Only one CC domain from the new subclade,
which is also the closest homolog to Sr50, has been assayed. Testing
more members across the newly identified subfamily will help to draw a
general conclusion about the new subclade.

\par\null

The manuscript proposes a possible diversification in signalling
capabilities, however, the chimera experiments of \cite{Jordan2011} are
not consistent with this hypothesis and suggest conserved signalling
capacities.

\par\null

\subsection*{(2)~~Comments on structural
predictions}

{\label{390222}}

The 21st residue of the MLA family CC domains is generally occupied by
an aspartate or a glutamate whereas it is a glycine in Sr33, and it is
suggested in the manuscript that this may account for the reported
differences in the structures---Sr33 was described as a four-helix
bundle by~\cite{Casey2016}, and MLA10 as a helix-loop-helix in an
obligate dimer by \cite{Maekawa2011}. The~\cite{Casey2016} paper has
shown that the CC domains of Sr33, MLA10 and Rx all maintain the same
oligomeric state and four-helix bundle fold in solution, and this is
supported by biophysical analyses of recombinantly produced protein. As
such, the current debate on the CC domain structure is not centred
around differences in their tertiary structures. What is unknown is
whether the MLA10 CC domain dimeric helix-loop-helix structure
represents an alternative quaternary conformation, for example a
post-activation conformation. To this end, the manuscript does not
address the ``alternative activation state hypothesis'' (discussed
by~\cite{27803318}), rather the text implies that the tertiary
structures of Sr33 and MLA10 are different. To support the statement
that Sr33 and MLA10 CC domains maintain different tertiary structures,
the authors applied secondary structure prediction with PSIPRED and
protein stability modelling with the STRUM web-server. However,
published biochemical and biophysical data demonstrating the structural
similarity of the Sr33 and MLA10 proteins in solution are not fully
considered.~

\par\null

Secondary structure predictions of the MLA10 and Sr33 CC domains were
stated to be performed with the first 40 amino acids ``for simplicity''.
This is problematic as secondary structure prediction using PSIPRED can
vary depending on the length of the sequence submitted. Indeed, when the
first 160 amino acids of MLA10 and Sr33 are submitted to PSIPRED, the
observed differences in the ``looped vs helical'' regions of the first
40 residues of Sr33 and MLA10 reported in this manuscript are no longer
apparent. Considering that the expression of the 1-160 region of the CC
domains (or equivalent) triggered cell death in planta (Figure 4a), we
believe this region to be more appropriate for secondary structure
predictions.

\par\null

It was hypothesized that the presence of the glycine at the 21st residue
in Sr33 is the determinant of the ``structural differences between MLA10
and Sr33'', and subsequently used the STRUM server (structure-based
prediction of protein stability changes upon single-point mutation), to
predict whether reciprocal mutations of polymorphisms between MLA10 and
Sr33 (Sr33 V20T and Sr33 G21E; MLA10 T20V and MLA10 E21G) would be
sufficient to destabilise the MLA10 and Sr33 structures, respectively.
The results of STRUM suggest the MLA10 T20V and MLA10 E21G would likely
destabilise the MLA10 structure, however the reciprocal mutations in
Sr33 would have no effect. There are several questions that this
analysis raises listed below.

\par\null

\begin{itemize}
\tightlist
\item
  What was the structure used to model the destabilisation? The original
  MLA10 structure (3QFL) could be compared to the four-helix bundle fold
  of the Rx and Sr33 CC domains. The small-angle X-ray scattering (SAXS)
  data for MLA10 CC domain published by \cite{Casey2016} indicates the
  MLA10 CC domain also likely forms a four-helix bundle in solution,
  therefore a better approach to this experiment would be to generate a
  homology model of MLA10 based on the Sr33 CC domain four-helix bundle
  and then assess the effects of the mutants on protein stability using
  STRUM. Additionally, it would be ideal if all the approaches taken to
  the predictions in the manuscript could be detailed in the materials
  and methods.
\item
  To simulate protein stability, it would be more appropriate to use the
  entire functional region of the protein instead of a non-functional
  shorter fragment, as it is likely that the missing residues could
  contribute to stability of the protein. Unfortunately, there is no
  structure of the active CC domain of MLA10 (a minimum of 142 residues)
  and as STRUM requires a structure to predict destabilisation caused by
  point mutations, it is not possible to determine the destabilising
  effect of the Sr33 V20T and Sr33 G21E mutants in a functional CC
  domain, even via homology modelling. Consequently, any stability
  prediction using either the current MLA10 or Sr33 CC domain structures
  are essentially not directly relevant to the minimal functional
  domain.~
\item
  Biochemical and biophysical analyses would be the more robust
  approaches than predictions of protein stability. These experiments
  are possible given that MLA10 CC domain can be purified in quantities
  sufficient for structural studies as in \cite{Maekawa2011}. For
  example, stability of MLA10 and Sr33 CC domain mutants could be
  analysed by circular dichroism (CD), 2D NMR, or by ThermoFluor. These
  experiments would be much more conclusive than prediction servers, and
  these data would significantly benefit the manuscript.
\item
  Finally, the MLA10 T20V and MLA10 E21G mutants appear to not have any
  effects on the cell death phenotype nor on the accumulation of these
  proteins in planta (Fig. 5). These observations are inconsistent with
  the hypothesis that the mutations destabilize the proteins. Further
  discussion of these observations would be beneficial.
\end{itemize}

\subsection*{}

{\label{485825}}

\subsection*{(3)~~Other comments}

{\label{485825}}

Figure S1 is a great positive control for the bioinformatics pipeline.

\par\null

It is not clear why the second clade is described as a SUB-family of MLA
given that ALL known MLA are in the other clade. The second clade is
better described as MLA-like or MLA sister clade.

\par\null

Some plants carry two (or even three) members of MLA. In these cases, do
they belong to the same or a different subclade? It was not clear in the
text and is worth commenting as this situation complicates allelic
analyses.

\par\null

Line 127: as there is not structural data available and to avoid
confusion, the text should state ``predicted to be located\ldots{}''

\par\null

Lines 142-143: The statement that RGH1/MLA family has been driven by
subfamily-specific functionalization to distinct pathogens is highly
speculative.~ Is there evidence for a second pathogen? Is possible that
this subfamily detects uncharacterized powdery mildew strains. This
contradicts lines 410-412 ``Whether subfamily 2 NLRs confer disease
resistance to avirulence genes present in yet uncharacterized Bgh
populations or other pathogens remains to be tested''.

\par\null

Line 257: ``Bootstrap not very high''. Perhaps include the bootstrap
number in brackets.

\par\null

Line 384. It is unclear how they can conclude from RNAseq data only that
``in wild barley Rgh1/Mla has undergone frequent gene duplication (Table
S1)''. Could these sequences be allelic?

\par\null

Lines 450-456. They cite \cite{Shen2007} as evidence that MLA-CC
functions by binding WRKY transcription factors and derepressing them.
Our understanding is that this model was drawn from an experiment with
the inappropriate avirulence effector.

\par\null

Figures 4 and 5: the loading control would be easier to distinguish when
showing the band corresponding to RuBisCO (55KDa).~\(\)

\par\null

\section*{Reviewers}

{\label{924286}}\par\null

Adam R. Bentham and Juan Carlos De la Concepcion, Department of
Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich,
UK.

\par\null

Sophien Kamoun. The Sainsbury Laboratory, Norwich Research Park,
Norwich, UK.

\par\null

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