Plants are continuously challenged by pathogens, affecting most staple crops compromising food security. They have evolved different mechanisms to counterattack pathogen infection, including the accumulation of pathogenesis-related (PR) proteins. These proteins have been implicated in active defense, and their overexpression has led to enhanced resistance in nuclear transgenic plants, although in many cases constitutive expression resulted in lesion-mimic phenotypes. We decided to evaluate plastid transformation as an alternative to overcome limitations observed for nuclear transgenic technologies. The advantages include the possibilities to express polycistronic RNAs, to obtain higher protein expression levels, and the impeded gene flow due to the maternal inheritance of the plastome. We transformed
Nicotiana tabacum
plastids to co-express the tobacco PR proteins AP24 and β-1,3-glucanase. Transplastomic tobacco lines were characterized and subsequently challenged with
Rhizoctonia solani
,
Peronospora hyoscyami
f.sp.
tabacina
and
Phytophthora nicotianae
. Results showed that transplastomic plants expressing AP24 and β-1,3-glucanase are resistant to
R
.
solani
in greenhouse conditions and, furthermore, they are protected against
P
.
hyoscyami
f.sp.
tabacina
and
P
.
nicotianae
in field conditions under high inoculum pressure. Our results suggest that plastid co- expression of PR proteins AP24 and β-1,3-glucanase resulted in enhanced resistance against filamentous pathogens.
Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that chloroplast expression of many proteins, such as human epidermal growth factor (hEGF), results hindered by post-transcriptional mechanisms. hEGF degradation has been related to the redox potential of the stroma and protein misfolding. To solve this problem, we proposed the redirection of hEGF into the thylakoid lumen where the environment could improve disulfide bonds formation stabilizing the functional conformation of the protein. We generated transplastomic tobacco plants targeting hEGF protein to the thylakoid lumen by adding a transit peptide (Str). Following this approach, we could detect thylakoid lumen-targeted hEGF by western blotting while stromal accumulation of hEGF remained undetectable. Southern blot analysis confirmed the integration of the transgene through homologous recombination into the plastome. Northern blot analysis showed similar levels of egf transcripts in the EGF and StrEGF lines. These results suggest that higher stability of the hEGF peptide in the thylakoid lumen is the primary cause of the increased accumulation of the recombinant protein observed in StrEGF lines. They also highlight the necessity of exploring different suborganellar destinations to improve the accumulation levels of a specific recombinant protein in plastids.
The pollen and pistil RALF peptides, along with multiple receptorlike kinases and leucine-rich repeat extensins, regulate pollen tube growth and the final burst within the ovule, where sperm cells are released for fertilisation to occur. This review introduces some new questions that arose about the regulation of this complex process.
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