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\begin{document}

\title{PREreview of bioRxiv article ``Cross-reactivity of a rice NLR immune
receptor to distinct effectors from the~ blast pathogen leads to partial
disease resistance''~~~~~~~~}



\author[1]{PMI Journal Club at MPIPZ}%
\affil[1]{PREreview}%


\vspace{-1em}



  
  \date{May 25, 2024}


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Review of: Cross-reactivity of a rice NLR immune receptor to distinct
effectors from the blast pathogen leads to partial disease resistance

Authors: Freya A. Varden, Hiromasa Saitoh, Kae Yoshino, Marina
Franceschetti, Sophien Kamoun, Ryohei Terauchi, Mark J. Banfield

DOI: http://dx.doi.org/10.1101/530675

\par\null

This manuscript examines the molecular mechanism of the recognition of
the ``mismatched'' \emph{Magnaporthe oryzae} (\emph{Mo)} effector AvrPia
by the rice CC-type NLR Pikp. The authors show that transient
co-expression of Pikp and AvrPia induces a weak cell death response in
\emph{N. benthamiana} (\emph{Nb}). In line with these results, a
Pikp-containing rice cultivar induces a weak immune response, whereas
the allelic Pikm cannot. Next, they show that the Pikp HMA domain
(Pikp-HMA) directly interacts with AvrPia \emph{in vitro}, while Pikm
cannot. X-ray crystallography showed that Pikp-HMA indeed directly binds
AvrPia in a manner reminiscent of the interaction between RGA5-HMA and
AvrCO39. This mode of interaction is in contrast to the previously
published AvrPikD Pikp-HMA structure. The authors propose that the
N-terminal extension of AvrPikD is responsible for the configuration of
the AvrPikD Pikp-HMA heterodimer and could enhance the AvrPia Pikp-HMA
interaction. However, addition of this N-terminal extension to AvrPia
did not enhance, but instead reduced, AvrPia-Pikp cell death output in
\emph{Nb}. Furthermore, this modified AvrPia version was also not
recognised by Pikm, which, like Pikp-AvrPikD, depends on the AvrPikD
N-terminal extension.

The finding that an integrated domain of an NLR is able to recognise two
effectors using two distinct interfaces is a novel finding and will be
of interest for the wider NLR biology community. Furthermore, these
findings have potential implications for the plant breeding industry.
However, we suggest that addressing the issues that we raise below will
significantly strengthen the authors' claims and the impact of their
study.

\par\null

\textbf{Major comments}

1. The authors use a cell death index to quantify the severity of the
cell death response in \emph{Nb} (Fig. 1 and 5). If the authors wish to
show these data we recommend to use stacked bar plots instead of box
plots. The use of box plots implies the data has a Gaussian
distribution, which clearly is not the case for many, if not all, of the
genotypes tested. Moreover, as the index only consists of a scale of 6
possible values, a Gaussian distribution cannot really be made.
Furthermore, it is unclear whether the differences depicted are
statistically significant. We would recommend to include this, and
clearly state the statistical test used in the figure legend. Most
importantly, we would advise to complement these data with ion leakage
measurements that are known for a quantifiable dose-response
relationship. Such physiological experiments will provide a more
quantitative readout and strengthen the claim that Pikp induces an
intermediate cell death response upon AvrPia perception. This is of
particular significance as ``partial disease resistance'' is one of the
important findings of this paper as stressed in the title. ~~

2. The authors show that the rice cultivar K60 containing Pikp induces a
partial immune response upon infection with \emph{Mo} Sasa2 (AvrPia).
This claim can be made based on the pictures shown in figure 2. However,
a quantification of the lesion size (area and/or lesion length) would be
desirable. Furthermore, it would strengthen the authors claim if they
would back up these observations with some physiological experiments,
\emph{e.g.} 1) measuring fungal growth by qPCR on fungal genomic DNA, 2)
Ion leakage, 3) staining rice leaves for ROS with DAB or H2DCFDA, 4)
measuring transcriptional outputs by qPCR on relevant marker genes, such
as \emph{RbohD}. Particularly comparing transcriptional outputs and ion
leakage over time in plants infected with \emph{Mo} AvrPikD and
\emph{Mo} AvrPia would be desirable. ~Not only will these data
strengthen the author's conclusions, they would yield insight into the
resistance pathway dynamics between \emph{Mo} AvrPikD- and \emph{Mo}
AvrPia-treated plants.

3. In figures 3 and 4 the authors clearly show that Pikp-HMA is able to
directly interact with AvrPia by gel filtration, SPR and x-ray
crystallography. Although these data are convincing, one aspect is still
missing. Since the interaction experiments are based on \emph{in vitro}
systems, it would strengthen the authors' claims if they would include
\emph{in vivo} interaction data. Yeast-2-Hybrid or \emph{in planta}
split-luciferase assays are two options the authors could explore.

4. The interaction data between Pikp and AvrPia are based on the
interaction of the Pikp-HMA and the effector AvrPia. It would strengthen
the authors' claims if they would confirm their interaction results by
performing interaction assays using the full length NLR instead of
merely the HMA domain. Yeast-2-Hybrid and split-luciferase would be
suitable techniques.

5. Given that the presented AvrPia Pikp-HMA structure as well as the
previously reported AvrPikD Pikp-HMA structure are based on HMA domains
lacking the adjacent NLR modules, how do the authors know that the
recognition of two distinct HMA interfaces occurs and is physiologically
relevant in the corresponding full-length NLRs? The authors do not show
a variant of Pikp (-HMA) that is unable to interact with AvrPia, even
though the authors discuss residues in Pikp-HMA that seem to be
responsible for the interaction, \emph{e.g.} PikpS202 \emph{} PikpD217,
and PikpDS204 (Fig. 4C). The authors could try to evaluate the effect of
mutating these three sites and determine whether this leads to a reduced
interaction between Pikp (-HMA) and AvrPia. Moreover, if this does
reduce binding, the authors could test whether these mutations also
affect Pikp outputs \emph{in planta, e.g.} by co-expressing the Pikp
variant with AvrPia in \emph{Nb} and quantifying signalling outputs by
ion leakage.

\par\null

\textbf{Minor comments}

1. Although the manuscript is well written, clear and succinct, in some
instances the authors refer to a previous finding, but do not supply the
according reference\emph{, e.g.} Introduction; paragraph 5: ``The
AvrPikC\ldots{} Pik NLR.'', and Introduction; paragraph 7:
``Despite\ldots{} on its own.''. Therefore, I would recommend the
authors to carefully go through the manuscript and make sure all
published data are appropriately referenced.

\par\null

Joram A. Dongus and the Plant Microbe Interactions Journal Club at MPIPZ

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